Objective To establish a superoxiaized model in vitro by using H₂O₂ in Hela cells and to observe the effect of H₂O₂ on the autophagy and apoptosis in Hela cells, and to investigate the relationship between H₂O₂ and endoplasmic reticulum stress(ERS) signal pathway. Methods 1 mmol·L^-1 H₂O₂ was employed to establish in vitro oxidative stress model.The experiment included control group, H₂O₂ 4 h group, H₂O₂ 8 h group, and H₂O₂ 12 h group;the Hela cells in various groups were treated with H₂O₂ for 0, 4, 8, and 12 h, respectively.The expression levels of apoptotic and autophagic proteins in the Hela cells in various groups were detected by Western blotting method. Results Compared with control group, the expression levels of Cleaved Caspase-3 in H₂O₂ groups were significantly increased (P<0.05), and the expression levels of Cleaved Caspase-4 were increased also(P<0.05).The expression levels of LC3-Ⅱ in H₂O₂ groups were also increased significantly compared with control group(P<0.05).The expression levels of GRP78 in H₂O₂ groups were increased compared with control group(P<0.05), and the expression levels of IRE1a(P<0.01) and p-JNK(P<0.05) were higher than those in control group. Conclusion H₂O₂ can induce the autophagy and apoptosis in Hela cells of in vitro oxidative stress model and its possible mechanism is related to ERS signal pathway.

Objective To determine the intervention effect of poly(ADP-ribose) polymerase 1 (PARP1) inhibition on the bleomycin(BML)-induced pulmonary fibrosis in anti-tumor therapies, and to further elucidate the mechanisms of anti-fibrosis role of PARP1 inhibition. Methods Forty eight female ICR mice were randomly divided into normal control (peritoneal injection of saline) and BLM groups (model groups) with doses of 5, 10 and 15 mg·kg^-1, respectively. BLM was peritoneally injected at day 1, 4, 8, 15, 18, 22 and 25. Sixty female ICR mice were randomly divided into normal control, 10 mg·kg^-1 BLM model group, and differeat doses(1, 5 and 10 mg·kg^-1) of PJ34-treated groups. BLM was administrated 20 min after intravenous injection of PARP1 inhibitor PJ34. In all above experiments, 3 mice were sacrificed at day 14, 21 and 28, the lungs were removed for histochemical analysis. HE staining was performed to detect the alveolar damage and Masson staining was performed to detects the collagen deposition. Moreover, A549 cells were exposed to BLM, and the effects of PARP1 inhibition on the expression of EMT biomarkers were detected by Real-time PCR. Results Compared with BLM model group, the alveolar damage and collagen deposition in the lung tissue of the mice in PJ34-treated group were effectively decreased. Compared with BLM group, the E-cadherin mRNA expression levels in A549 cells of the mice in PJ34-treated group was increased (P<0.05), and the expression levels of α-SMA and TGF-β mRNA were decreased(P<0.05 or P<0.01). Conclusion PARP inhibition can affect the expressions of EMT marker genes, and alleviate the BLM-induced pulmonary fibrosis, suggesting PARP1 inhibitors can be utilized to prevent the pulmonary fibrosis in the process of cancer therapy.

Objective To investigate the expression of ATP binding cassette transporter A1(ABCA1) in serum obtained from peripheral blood of the patients with preeclampsia, and to clarify its effects on the injury of endothelial cells and possible mechanisms. Methods 40 cases of mild or severe preeclampsia women and 20 cases of normal pregnancy women were randomly selected, whose period of hospitalization and gestational age had no significant differences.The expression levels of serum ABCA1 and oxidized low-density lipoprotein (OX-LDL) in peripheral vein blood were determined by using ELISA.The primary isolated and cultured umbilical vein endothelial cells (HUVECs) were obtained from the three groups.The function of endothelial cells was tested on the basis of following aspects:monolayer barrier function was evaluated by transferring fluorescently-labeled BSA across the monolayer HUVECs;the cell proliferation was measured by using MTT assay;the levels of secretory nitric oxide(NO)was assayed by nitrate reductase assay. Results The expression levels of ABCA1 protein in serum of the patients in mild or severe preeclampsia groups were significantly lower, whereas the expression levels of OX-LDL and the ratios of OX-LDL/ABCA1 were significantly higher than those in normal pregnancy group(P<0.05).There was obviously negative correlation between the levels of ABCA1 and OX-LDL in the patients with preeclampsia(r=-0.681, P<0.05).Compared with normal pregnancy group, the BSA levels across the monolayer HUVECs of the patients in preeclampsia groups were significantly increased(P<0.05), the proliferation ability of HUVECs was markedly decreased(P<0.05), and the level of secretory NO was significantly reduced(P<0.05).And the more severe the disease, the more significant these changes.The ratio of serum OX-LDL/ABCA1 was positively correlated with the BSA level (r= 0.791, P<0.05), and was negatively correlated with the endothelial cell proliferation level(r=-0.812, P<0.05), and was negatively correlated with the endothelial NO level(r=-0.635, P<0.05) of the patients in preeclampsia groups. Conclusion The expression level of ABCA1 protein in serum of the patients with preeclampsia is decreased which can cause the oxidative stress and OX-LDL/ABCA1 expression imbalance.It is closely related to systemic vascular endothelial cell damage which may be one of the important mechanisms involved in the development of preeclampsia.

Objective To explore the cytotoxicity of silica nanoparticles(SiNPs) on the vascular endothelial cells and possible mechanism, and to provide reference for the study on the toxic effects and safety evaluation of SiNPs. Methods The in vitro cultured human umbilical vein endothelial cells (HUVECs) were randomly divided into control and SiNPs exposure groups at the concentrations of 12.5, 25.0, 50.0, and 100.0 mg·L^-1, respectively.Transmission electron microscope (TEM) was used to determine the particle size, morphology and dispersion of SiNPs.After 24 h exposure, inductively coupled plasma atomic emission spectroscopy (ICP-AES) was used for the measurement of intracellular silicon level, MTT assay for the determination of cell viability, lactate dehydrogenase (LDH) release assay for membrane integrity, laser confocal microscope with DCFH-DA fluorescence probe for intracellular reactive oxygens pecies (ROS) level, DAPI staining under fluorescence microscope for the observation of nuclear morphology, flow cytometry (FCM) with Annexin Ⅴ and PI double staining for the measurement of apoptosis, and Rhodamine 123 kits for mitochondrial membrane potential (MMP). Results The TEM Results showed that SiNPs were mostly spherical with uniform size and well dispersed.The average particle size was (57.66±7.30) nm calculated by Image J software.Compared with control group, the cell viabilities in SiNPs exposure groups were decreased(P<0.05), while the intracellular silicon levels and LDH activities in medium were increased (P<0.05);the ROS levels were increased, the MMP was declined, and the apoptosis appeared.All the cell effects mentioned above were enlarged in a dose-dependent manner. Conclusion SiNPs have toxicities to vascular endothelial cells, which could induce ROS generation and oxidative stress, further can do damage to cellular membrane and mitochondria, and thus lead to apoptosis eventually.

Objective To explore the effect of CD147 on the invasion of prostate cancer LNCaP-AI cells and its possible mechanism in vitro, and to provide basis for research on prostate cancer invasion. Methods The shRNA vector targeting CD147 gene and negative control vector were transfected into LNCaP-AI cells by Lipofectamine 2000, respectively.The stable cell lines were obtained after G418 screening and divided into AI/shCD147 group (experiment group) and AI/Scramble group (control group), respectively.Western blotting method was employed to detect the expression of CD147 protein in the cells in two groups.Transwell invasion assay was used to evaluate the invasion ability of LNCaP-AI cells in vitro.The level of matrix metalloproteinase-2 (MMP-2) was determined by Western blotting method. Results Compared with AI/Scramble group, the CD147 protein in the LNCaP-AI cells in AI/shCD147 group was significantly decreased.The Transwell invasion assay showed that the A value of LNCaP-AI cells in AI/shCD147 group (1.43±0.2) was significantly lower than that in AI/Scramble group (2.08±0.3) (P<0.05).Compared with AI/Scramble group (0.85±0.02), the expression of MMP-2 protein in AI/shCD147 group (0.46±0.08) was significantly decreased (P<0.05). Conclusion CD147 can promote the invasion ability of LNCaP-AI cells in vitro through inducing the MMP-2 secretion.It is demonstrated that targeting CD147 could potentially be a new approach for prostate cancer gene therapy.

Objective To obseve the rapair effect of hepatic progenitor cells (HPCs) transplantation on the liver of mouse models of acute liver failure induced with different concentrations of carbon tetrachloride (CCl₄), and to construct one ideal mouse model of acute liver failure for evaluation on the efficacy of HPCs transplantation. Methods 96 ICR mice were randomly divided into nine groups: blank control group, negative control groups (0.1% CCl₄ group, 0.5% CCl₄ group, 2.0% CCl₄ group and 10.0% CCl₄ group) and experimental groups (0.1% CCl₄ + HPCs group, 0.5% CCl₄ + HPCs group, 2.0% CCl₄ + HPCs group and 10.0% CCl₄ + HPCs group).Normal saline was given to the mice in blank control group by intragastric administration, and the mice in negative control groups and experimental groups were administrated with different concentrations of CCl₄.One day later, the mice in experimental groups were injected with HPCs from spleen, and the mice in negative control groups were given the same dose of normal saline.14 d after the injection of HPCs, the survival rate, liver index, the activities of serum alanine aminotransferase(ALT) and aspartate aminotransferase(AST), the histopathological changes, the distribution of exogenous hepatocytes and the expression levels of cytokeratin 18(CK18) and albumin (ALB) were detected. Results Compared with blank control group, the survival rates, liver indexes and the activities of serum ALT and AST in 0.1% CCl₄ group and 0.5% CCl₄ group had no significant changes(P>0.05);the effective treatment of HPCs transplantation was not observed because of self-repairing.Compared with 2.0% CCl₄ model group, the activities of serum ALT and AST and liver index in 2.0% CCl₄ + HPCs group were significantly decreased(P<0.01), and the survival rate was increased(P<0.01);the liver cell regeneration and a large number of exogenous hepatocytes were found by pathological detection in 2% CCl₄ + HPCs group;the therapy of HPCs transplantation was effective.All mice died in 2 d in 10.0% CCl₄ group and 10.0% CCl₄ + HPCs group, and the therapeutic efficacy of HPCs was not observed. Conclusion HPCs transplantation can increase the survival rate of the model mice of acute liver failure, improve the activities of ALT, and AST and repair the hepatic injury;HPCs transplantation treatment is effective on the mouse model of 2.0% CCl₄-induced acute liver failure.

Objective To discuss the effect of As₂O₃ on the apoptosis of HepG₂ cells via Notch signal pathway, and to clarify its mechanism. Methods The HepG₂ cells cultured in vitro were divided into blank group, As₂O₃ group, MW167 group and combination group.The HepG₂ cells were treated with As₂O₃ and MW167 at different concentrations for 24, 48 and 72 h, and the inhibitory rates of proliferations of the HepG₂ cells were detected by MTT method.The HepG₂ cells in various groups were intervened by MW167 and As₂O₃ at non-cytotoxic doses (As₂O₃ 0.25 mg·L^-1, MW167 10 μmol·L^-1) based on The Results of MTT for 48 h, then the apoptotic rate was tested by flow cytometry;RT-PCR and Western blotting Methods were employed to detect the expressions of Notch-1, Bcl-2 and Caspase-3 mRNA and protein. Results The HepG₂ cells treated with both As₂O₃ and MW167 at different concentrations could inhibit the cell proliferation and showed a dose-effect relationship.For the HepG₂ cells in different groups treated with MW167 group and As₂O₃ group at non-cytotoxic doses for 48 h, compared with blank group, the apoptotic rates in As₂O₃ group, MW167 group and combination group were increased(P<0.05), especially in combination group; the expression levels of Bcl-2, Notch-1 mRNA and protein in As₂O₃ group, MW167 group and combination group were lower than those in blank group(P<0.05), especially in combination group;compared with blank group, the expressions levels of Caspase-3 mRNA and protein in As₂O₃ group, MW167 group and combination group were increased(P<0.05), especially in combination group. Conclusion As₂O₃ can induce the apoptosis of hepatocelular carcinoma HepG₂ cells and the mechanism may be related to the down-regulation of Bcl-2 and Notch-1 and up-regulation of Caspase-3 mRNA and protein through Notch signal pathway.

Objective To investigate the effect of gambogic acid lysinate (GAL) on the proliferation and apoptosis of human breast cancer MCF-7 cells and its mechanism, and to provide theoretical foundation for clinical trial and use of GAL against breast cancer. Methods The human breast cancer MCF-7 cells in logarithm growth phase were selected and randomly divided into blank control group, different doses (0.25, 0.50, 1.00, 2.00, 4.00, 8.00 μmol·L^-1) of GAL groups, and different doses(0.25, 0.50, 1.00, 2.00, 4.00, 8.00 μmol·L^-1) of gambogic acid groups.After the MCF-7 cells were treated with GAL or gambogic acid for 48 h, MTT assay was used to detect the proliferation activities of MCF-7 cells.After the MCF-7 cells in logarithm growth phase were treated with 2 μmol·L^-1 GAL for different time (0, 6, 12, 24, 36, 48, 60 h), MTT assay was also used to detect the proliferation activities of MCF-7 cells in various groups.The flow cytometry method and Hoechst 33258 staining were used to analyze the apoptosis of MCF-7 cells in various groups. The expression levels of apoptosis-associated proteins were detected by Western blotting method. Results After 48 h cell culture, the cell viabilities in GAL groups were lower than those in control group (P< 0.05) and the cell viability was inhibited in a dose-dependent manner (P<0.05).After the MCF-7 cells were treated by 2 μmol·L^-1 GAL for different time, the cell proliferation was inhibited in a time-dependent manner (P<0.05).The flow cytometry method and Hoechst 33258 staining Results showed that the apoptotic rates of MCF-7 cells in GAL groups were higher than those in control group (P<0.05).The expressions levels of SIRT1 in GAL groups were significantly lower than that in control group (P<0.05), while the expression amounts of cleaved caspase-3 were higher than that in control group (P<0.05). Conclusion GAL can induce the apoptosis of breast cancer MCF-7 cells by inhibiting the expression level of SIRT1 and up-regulating the expression amount of cleaved caspase-3.

Objective To investigate the therapeutic effect of rhein lysinate (RHL) on the liver fibrosis in the cholestatic model rats, and to expound the molecular mechanisms of RHL in treatment of liver fibrosis in the cholestatic rats. Methods 35 rats were divided into control group, model group, 35 and 70 mg·kg^-1 RHL treatment groups, and lysine group(n=7).The rat models of cholestatic hepatic fibrosis were established by bile duct ligation (BDL) method.The activities of serum aspartate aminotransferase (AST), alanine aminotransferase (ALT) and the levels of total bile acid (TBA), total bilirubin (TBIL) were detected by using an automatic biochemical analyzer;the pathological changes of liver tissue of the rats in various groups were observed by HE staining;the content of fiber in liver tissue was observed by Masson's trichrome stain;the level of hydroxyproline (Hyp) was detected by microplate reader according to the instruction of respective kits;the expression of α-smooth muscleactin (α-SMA) was detected by immunohistochemical staining;the expression level of α-SMA was detected by Western blotting method. Results Compared with control group, the ratio of liver weight to body weight and the activities of AST and ALT, the levels of TBA and TBIL in serum of the rats in model group were increased (P<0.05);the hepatic lobule structure of hepatic tissue was destroyed, and the fibrous tissue hyperplasia was found, the Hyp level was also increased(P<0.05);the immunohistochemical staining Results showed that α-SMA expressed in cell membrane and cytoplasm, and the Western blotting Results showed that the expression level of α-SMA protein was increased(P<0.05). Compared with model group, the ratio of liver weight to body weight and the activities of AST, ALT and the levels of TBA, TBIL in serum in 35 and 70 mg·kg^-1 RHL groups were significantly decreased(P<0.05);the liver histopathological examination found that the fibers of the rats in 35 and 70 mg·kg^-1 RHL groups were reduced, and the levels of Hyp were also decreased(P<0.05); the immunohistochemistry and Western blotting Results showed that the levels of α-SMA protein were reduced(P<0.05). Conclusion The hepatic protection of RHL is correlated with the inhibition of the activation of hepatic stellate cells, fibrosis and fiber accumulation of liver tissue.It is concluded that RHL would be a therapeutic drug for the patients with cholestatic liver fibrosis.

Objective To detect the inhibitory effects of N-acetyl-cysteine(NAC)on the increasing of reactive oxygen species(ROS)level induced by SiO₂ nanoparticles, and to clarify the mechanism of cytotoxicity induced by SiO₂ nanoparticles. Methods The 68 nm SiO₂ nanoparticles were selected and the in vitro cultured human normal liver L-02 cells were used as cell model.The L-02 cells were divided into control group, different concentrations of NAC(1, 2, 5, 10 mmol·L^-1)pretreatment groups, different concentrations of SiO₂ nanoparticle(10, 20, 50, 100 mg·L^-1)groups and SiO₂ nanoparticle exposure with NAC pretreatment groups.MTT assay was applied to measure the viabilities of the L-02 cells in NAC pretreatment groups with and without SiO₂ nanoparticle exposure.The morphology of L-02 cells pretreated with NAC followed by SiO₂ nanoparticles exposure was observed by inverted microscope.The intracellular ROS levels were measured by fluorescence microplate reader to evaluate NAC effects towards different concentrations of SiO₂ nanoparticles. Results The cell viabilities in NAC pretreatment groups were elevated, and peaked in 5 mmol·L^-1 group.However, compared with control group, the NAC did not increase the cell viability significantly(P>0.05).Compared with SiO₂ nanoparticle exposure group without NAC, the cell viabilities in NAC pretreatment groups were increased(P<0.05).The image of NAC pretreatment group showed higher cell number and more distinct cell boundary compared with SiO₂ nanoparticle group without NAC.With the increase of SiO₂ nanoparticles concentration, the intracellular ROS levels were increased in a dose-dependent manner.Compared with ocntrol group, the ROS levels in 50 and 100 mg·L^-1 SiO₂ nanoparticle groups were increased significantly(P<0.05).The ROS levels in SiO₂ nanoparticles exposure with NAC pretreatment groups were decreased compared with SiO₂ nanoparticles exposure groups at the same concentration(P<0.05). Conclusion NAC can inhibit the decrease of cell viability and the increase of ROS level induced by SiO₂ nanoparticles to some extent.The intracellular ROS level increase is one of the mechanisms of SiO₂ nanoparticles-induced cytotoxicity.

Objective To investigate the influence of velvet antler polypeptides (VAP) in the expressions of bone morphogenetic protein-2 (BMP-2) and runt-related transcription factor 2 (Runx2) gene and protein in human bone marrow mesenchymalstem cells (hBMSCs), and to clarify the mechanism of VAP in bone synthesis and bone differentiation direction. Methods The cultured hBMSCs were divided into control group and 5, 10, 15, 20 g ·L^-1 VAP groups.By detecting the alkaline phosphatase (ALP) activities in hBMSCs in various groups, the optimal concentration of the drug in vitro was selected;CCK-8 assay was used to detect the proliferation of hBMSCs in control group and 10 g·L^-1 VAP group;the BMP-2 and Runx2 gene and protein expressions in control group and 10 g·L^-1 VAP group were determined with fluorescence quantitative PCR and Western blotting method. Results The ALP activities in VAP groups were higher than that in control group, and the ALP activity in 10 g ·L^-1 VAP group was the highest (P<0.01).The CCK-8 assay Results showed that the proliferation rate of the hBMSCs in 10 g ·L^-1 VAP group was higher than that in control group (P<0.05), the VAP-induced proliferation of cultured hBMSCs was the highest at the 9th day, then went into the plateau and began to decline;the quantitative PCR and Western blotting Results showed that the expression levels BMP-2 and Runx2 gene and protein in the hBMSCs in 10 g ·L^-1 VAP group were significantly higher than those in control group, and the differences were statistically significant(P<0.05). Conclusion VAP can improve the ALP activity in hBMSCs and promote the proliferation and differentiation of hBMSCs, and increase the expressions of BMP-2 gene and its Runx2 gene and protein, which may be one of the molecular regulation mechanisms of VAP for bone formation and bone metabolism of hBMSCs.

Objective To investigate whether N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) can inhibit the development of silicosis through blocking PDGF/ROCK pathway. Methods SiO₂ were douched in bronchial tube of the rats to make the silicotic models.Sixty Wistar rats were randomly divided into model control 4 weeks group, model control 8 weeks group, silicosic model 4 weeks group, silicosic model 8 weeks group, AcSDKP post-treatment group and AcSDKP pre-treatment group (n=10).The expression and distribution of phospho-PDGFR-β in lung tissue of the rats in various groups were observed by immunocytochemistry.The expressions of α-smooth muscle actin (α-SMA), PDGFR-β, phospho-PDGFR-β, ROCK, Ⅰ collagen and Ⅲ collagen in lung tissue of the rats in various groups were detected by Western blotting method. Results Compared with the corresponding control groups, the expression levels of α-SMA, PDGFR-β, phospho-PDGFR-β, ROCK, type Ⅰcollagen and type Ⅲ collagen in lung tissue of the rats in silicosic model groups were increased significantly(P<0.05).In addition, in silicosic model groups, there were more phospho-PDGFR-β positive cells distributed in the silicon nodules and interstitial fibrosis area detected by immunocytochemistry.However, these effects were inhibited in AcSDKP post-treatment group and AcSDKP pre-treatment group (P<0.05).The Results of immunohistochemical staining showed the expression of phospho-PDGFR-β protein was obviously decreased in AcSDKP post-treatment group and AcSDKP pre-treatment group. Conclusion PDGF/ROCK pathway may play an important role in the development of silicosis by promoting myofibroblast differentiation and further secreting more collgen; however, these effects may be inhibited by AcSDKP.

Objective To investigate the expression levels of collagenⅠ (ColⅠ) and osteocalcin (OC) in the bone tissue around the arg-gly-asp(RGD) peptide coated micro-screw implants in the Beagle's maxilla at different healing time, and to clarify the promotion of RGD-coated surface on the osseointegration of micro-screw implants. Methods Six male adult Beagles were included in this study.Thirty-six micro-implants were randomly divided into two group:experimental group (with RGD) and control group (without RGD)(n=18).A micro implant was inserted in any one of the maxilla implantation regions(furcation zone of second premolar, third premolar, forth premolar) respectively at the 0 week, 4 weeks, and 6 weeeks after experiments by the self-contrast methods, one side was used as the experimental group and another side as the control group.At the 8th week, all animals were sacrificed and the bone specimens were prepared for HE and immunohistochemical staining, and the expression levels of ColⅠand OC in the bone tissue around the micro-implant were detected. Results The Results of HE staining showed that there were more osteogenesis activity both in experimental group and control group at the 2nd and 4th weeks, while lamellar bone could be seen in two groups at the 8th week.The expression levels of ColⅠand OC in the bone tissue around the micro-screw implants in experimental group were obviously higher than those in control group at the 2nd and 4th weeks(P<0.05);but there was no significant difference at the 8th week between two groups(P>0.05).The expression levels of ColⅠin the bone tissue around the micro-screw implants in experimental group were significantly higher at the 4th week than those at the 2nd and 8th weeks(P<0.05), but there was statistical difference only between the 2nd and 8th weeks in control group(P<0.05).The OC expression levels in two groups were increased progressively at the 2nd week and the 4th week, and reached the highest level at the 8th week, and they were obviously higher at the 8th week than those at the 2nd week(P<0.05).When compared with the 2nd week, the OC expression levels in the bone tissue around the micro-screw implants in experimental group at the 4th week had the statistical difference (P<0.05), while no significant difference was found in control group(P>0.05). Conclusion The RGD-coated surface can increase the expressions of ColⅠand OC in the bone tissue around the micro-implants, and it plays a key role in improving the initial osseointegration of micro-implants.

Objective To study the stabilities of recombinant human fibroblast growth factor 21 (rhFGF21) deposited at different conditions, and to elucidate the influence of temperature, buffer solutions and repeated freeze thawing in the stability of rhFGF21. Methods rhFGF21 was deposited at different conditions such as temperatures (-20℃, 4℃, 25℃), buffer solutions (acetate sodium acetate buffer pH 4.0 and pH 5.0, citric acid-sodium citrate buffer pH 5.0 and pH 6.0, disodium hydrogen phosphate and sodium dihydrogen phosphate buffer pH 6.0, pH 6.5and pH 7.0, Tris - HCl buffer pH 7.5 and pH8.0) and repeated freeze thawing, respectively.The purity of rhFGF21 was determined by HPLC, and the appearance was also observed. Results All properties of rhFGF21 were not altered during the observation period at -20℃ and 4℃;the purity of rhFGF21 deposied for 14 d was reduced to (15.20±0.14)% compared with 0 d(100%±0.00%) at 25℃(P<0.05).The acetate sodium acetate buffer pH 4.0 and pH 5.0 and citric acid sodium citrate buffer pH 5.0 and pH 6.0 were turbidity under the condition of 25℃ for 7 d;the disodium hydrogen phosphate and sodium dihydrogen phosphate buffer pH 6.5 and pH 7.0, and Tris-HCl buffer pH 7.5 and pH 8.0 were clear transparent under the condition of 25℃ for 7 d. The purity of rhFGF21 had significant change after repeated with freeze thawing (P<0.05). Conclusion rhFGF21 is stable when deposited at low temperature and pH 6.5-8.0 neutral conditions.High temperature, acidic and basic conditions are harmful for the stability of rhFGF21.It is profit for rhFGF21 stability in sodium dihydrogen phosphate buffer.There is impact on rhFGF21 stability after freeze thrawing repeatedly.

Objective To study the effects of arg-gly-Asp (RGD) peptides coated porous tantalum(Ta) on the adhesion, proliferation and secretion of chondrocytes, and to investigate the feasibility of RGD/chondrocyte/porous tantalum composites for chondral defect repair and regeneration. Methods RGD peptides with different concentrations were loaded to the porous tantalum(Ta) surface by physical absorption and divided into 7 groups:Ta-RGD, Ta-ColⅡ, Ta-RGD/ColⅡ0.2 g·L^-1, Ta-RGD/ColⅡ 1.0 g·L^-1, Ta-RGD/ColⅡ5.0 g·L^-1, Ta-RGD/ColⅡ10.0 g·L^-1 and Pure Ta groups.The articular chondrocytes from immature rabbits were cultured and the 2rd generation chondrocytes were seeded onto porous tantalum.The morphology of RGD/chondrocyte/porous tantalum composites was observed by scanning electron microscopy(SEM).The adhesion rate of chondrocytes was detected by precipitation process, the proliferation level of chondrocytes was measured by MTT assay, and the changes of hydroxyproline level of chondrocytes were checked. Results The adhesion rate of chondrocytes in the porous tantalum modified with RGD peptides was higher than that in the unmodified porous tantalum(P<0.05).The best cell adhesion effect was gained in Ta-RGD/ColⅡ5.0 g·L^-1 group at 4 h and there were significant differences between various groups (P<0.05).The SEM found that the chondrocytes growed best on the surface and inner of porous tantalum and secreted extracellular matrix.The Results of MTT showed that the proliferation level of chondrocytes in the porous tantalum modified with RGD peptides was higher than that in the unmodified porous tantalum(P<0.05) and the best effect was gained in Ta-RGD/ColⅡ1.0 g·L^-1 group at 13 d.The determination of hydroxyproline showed that the hydroxyproline level of chondrocytes in Ta-RGD/ColⅡ1.0 g·L^-1 group was significantly higher than those in other groups(P<0.05). Conclusion The porous tantalum modified with RGD peptides is advantageous to enhance chondrocyte adhesion and proliferation on the surface and inner of porous tantalum and promots the secretion of chondrocytes.

Objective To observe the changes of number of the CD4⁺CD25⁺regulatory T cells, the expression level of CTLA-4 mRNA and the level of serum TGF-β, and to clarify the effect of inactivated Bacillus Calmette-Guerin vaccine(BCG) on the number of the CD4⁺CD25⁺regulatory T cells and CTLA-4 mRNA expression. Methods Thirty Sprague-Dawley (SD) rats were randomly divided into control group, model group and BCG group (n=10).The rats in model group and BCG group were sensitized by injecting intraperitioneally with OVA for 3 weeks;On the 22th day, they were challenged with 2% OVA for 6 weeks to built the chronic rasthma models.The rats in control group were sensitized and challenged with normal saline.The rats in BCG group were intradermally injected with inactivated BCG 3 d before sensitizing lasting 9 weeks.The airway resistances of the rats in various groups were assessed after intubation.The number of CD4⁺CD25⁺regulatory T cells and the expression level of CTLA-4mRNA were detected.The levels of serum TGF-β were detected by ELISA.The number of cells and the percentage of eosinophil(EOS) in bronchial alveolar lavage fluid (BALF) of the rats in various groups were detected. Results Compared with control group, the total number of cells and the percentage of EOS in BALF of the rats in model group were decreased (P<0.05);compared with model group, the total number of cells and the percentage of EOS in BALF of the rats in BCG group were increased(P<0.05).Compared with control group, the airway resistances of the rats in model group was increased when the concentration of histamine was 0.32 g·L^-1;compared with model group, the airway resistance of the rats in BCG group was decreased when the concentration of histamine was 0.64 g·L^-1.Compared with control group, the number of CD4⁺CD25⁺ regulatory T cells, the expression level of CTLA-4mRNA and the level of serum TGF-β of the rats in model group were decreased (P<0.05);compared with model group, CD4⁺CD25⁺ regulatory T cells, the expression level of CTLA-4 mRNA and the level of serum TGF-β of the rats in BCG group were increased (P<0.05). Conclusion The inactivated BCG can improve the airway inflammation and airway hyperresponsiveness in the asthmatic rats, and the mechanisms are correlated with the the number and the function of CD4⁺CD25⁺ regulatory T cells.

Objective To investigate the inhibitroy effect of bis(maltolato)oxovanadium(Ⅳ) (BMOV) on endoplasmic reticulum stress(ERS)-induced apoptosis of cardiomyocytes of the rats with diabetes mellitus (DM) and possible mechanisms, and to provide a theoretical basis for the clinical study on diabetic cardiomyopathy(DCM). Methods 35 male Wistar rats were selected, among them 10 rats were randomly selected and used as control group, and they were admistrated intraperitoneally with citric acid buffer (55 mg·kg^-1) and normal drinking water for 4 weeks.The remaining 25 rats were admistrated intraperitoneally with streptozotocin (STZ).1 week later, the rats with the level of fasting blood glucose greater than or equal to 13.3 mmol·L^-1 were used as DM models. Finally 20 DM rats were randomly divided into DM group and BMOV group (n=10).The rats in DM group were administered with normal drinking water for 3 weeks.The rats in BMOV group were administered with BMOV in drinking water for 3 weeks.The myocardium tissue was taken for TUNEL staining to observe the apoptosis;Western blotting method was used to detect the levels of glucose-regulated protein 78 (GRP78), CCCAAT/enhancer binding homologous protein (CHOP), X-box binding protein 1(XBP-1);qPCR was used to dectect the XBP-1 mRNA expression levels in myocardium tissue of the rats in various groups. Results The TUNEL staining Results showed that the apoptotic cells in cyocardium tissue of the rats in control group were few, in control group the apoptotic index(AI) was 0.80 + 0.63;compared with control group, the apoptosis of myocardial cells in DM group was significantly increased, and the AI was increased (P<0.05);compared with DM group, the apoptosis of myocardial cells in BMOV group was significantly decreased, and the AI was decreased (P<0.05).The Western blotting Results showed that the expression levels of GRP78, CHOP.and XBP-1 of the rats in DM group were significantly higher than those in control group(P<0.05);and the expression levels of GRP78, CHOP and XBP-1 of the rats in BMOV group were lower than those in DM group(P<0.05).The qPCR Results showed that the XBP-1 mRNA expression level of the rats in DM group was higher than that in control group(P<0.05), and the XBP-1 mRNA expression level of the rats in BMOV group was lower than that in DM group(P<0.05). Conclusion BMOV has protective effects on the myocardial cells of the DM rats, and its mechanism may be related to inhibition of ERS and apoptosis of myocardial cells induced by ERS.

Objective To investigate the effects of BeiMuGuaLou powder on the expression of angiotensin Ⅱ(AngⅡ) in thelung tissue of the rats with chronic obstructive pulmonary disease(COPD), and to study the protective effect of BeiMuGuaLou powder on lung tissue and its mechanism. Methods Thirty male SD rats were randomly divided into control group, COPD group and BeiMuGuaLou powder group, 10 rats in each group.The model of COPD was established by smoking combined with endotracheal injection of lipopolysaccharide(LPS) for 30 d.On the 8th day of modeling, the rats in control and COPD groups were lavaged with normal saline(20 mL·kg^-1).The rats in BeiMuGuaLou powder group were lavaged with BeiMuGuaLou powder(20 mL·kg^-1) for 22 d.The expression levels of AngⅡ in the lung tissue of the rats in various groups were determined by immunohistochemistry and Western blotting methods. Results Compared with control group, the rats in model group appeared dyspnea, cough, shortness of breath, apathia, spit saliva, yellow hair, yellow teeth, and other symptoms and signs.Compared with control group, the expression level of AngⅡ in lung tissue of the rats in model group was significantly increased(P<0.05);compared with model group, the expression level of AngⅡ in lung tissue of the rats in BeiMuGuaLou powder group was significantly reduced (P<0.05). Conclusion BeiMuGuaLou powder can resist the cell inflammatory reaction induced by AngⅡ, and obviously improve the asthma and cough, shortness of breath and other symptoms of the COPD rats.

Objective To investigate the protective effects of Schisandra Chinensis lignans (SCL) on the oxidative stress injury of PC12 cells induced by hydrogen peroxide and influence in NF-κB/iNOS/NO signaling pathway, and to explore the mechanisms. Methods The PC12 cells were divided into control group, model group(H₂O₂ 200 μmol·L^-1), high dose of SCL group(SCL 30 mg·L^-1+ H₂O₂ 200 μmol·L^-1, SCL₁ group)and low dose of SCL group(SCL 10 mg·L^-1+ H₂O₂ 200 μmol·L^-1, SCL₂ group).The PC12 cells were untreated (control group) or treated with H₂O₂ (200 μmol·L^-1) for 6 h (model group).The PC12 cells in SCL₁ and SCL₂ group were firstly pretreated with different concentrations of SCL for 2 h, and then continuously with SCL and exposed to 200 μmol·L^-1 H₂O₂ for 6 h at the same time.The survival rate of PC12 cells was evaluated by MTT assay.The activities of lactate dehydrogenase (LDH), superoxide dismutase (SOD) and the levels of malondialdehyde (MDA) and nitric oxide (NO) were detected by microplate reader.The fluorescence intensity of reactive oxygen species (ROS) was assessed with flow cytometry.Immunohistochemistry and Western blotting Methods were used to detect the protein expressions of inducible nitric oxide synthase (iNOS) and nuclear factor kappa B P65 (NF-κB P65). Results Compared with control group, the survival rates of PC12 cells in model group were significantly decreased (P<0.01);the LDH activities in the supernatant were increased (P<0.01), the SOD activities were decreased(P<0.01), the MDA and NO levels were increased (P<0.01), the cell ROS level was increased (P<0.01), the number of positive cells of NF-κB were increased (P<0.01), and the expression levels of iNOS and NF-κB P65 protein were increased (P<0.01). Compared with model group, the survival rates of PC12 cells in SCL₁ and SCL₂ groups were increased (P<0.05 or P<0.01), the LDH activities in the supernatant were decreased (P<0.05 or P<0.01), the SOD activities were increased (P<0.01), the MDA and NO levels were decreased (P<0.05 or P<0.01), the cell ROS levels was decreased (P<0.01), the number of positive cells of NF-κB was decreased (P<0.05 or P<0.01), and the expression levels of iNOS and NF-κB P65 protein were decreased (P<0.05). Conclusion SCL could protect the oxidative stress injury of PC12 cells, its mechanism may be related to inhibiting the NF-κB/iNOS/NO signaling pathway, reducing the free radical generation and alleviating lipid peroxidation damage.

Objective To observe the changes of the expressions of tumor necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1β) in the brainstem and the number of inflammatory cells in brainstem subarachnoid of the rat models with migraine, and to explore the inhibitory effect of Zhengqingfengtongning on aseptic neurogenic inflammation(ANI) of the rats with migraine. Methods 60 healthy Wistar rats were randomly divided into blank control group, model group, sumatriptan(6 mg·kg^-1·d^-1) group, low dose of Zhengqingfengtongning(100 mg·kg^-1·d^-1) group, middle dose of Zhengqingfengtongning(150 mg·kg^-1·d^-1) group and high dose of Zhengqingfengtongning(200 mg·kg^-1·d^-1)group(n=10).The migraine rat model was established by subcutaneous administration of nitroglycerin.The brain and mater of the rats were decapitated 4 h after medical intervention.The expressions of TNF-α and IL-1β in the rat brainstem were evaluated by immunohistochemistry, and the number of TNF-α and IL-1β positive cells under microscope was counted.HE staining was used to observe the changes of the number of inflammatory cells in the brainstem subarachnoid of the rats and the number of inflammatory cells was counted under light microscope of per unit area subarachnoid space(in a view for unit). Results Compared with blank control group, the number of TNF-α and IL-1β expression positive cells in the brainstem of the rats in model group was increased significantly, and the differences were statistically significant(P<0.01);compared with model group, the number of TNF-α and IL-1β expression positive cells in the brainstem of the rats in middle and high doses of Zhengqingfengtongning groups and sumatriptan group was significantly decreased, the differences were statistically significant(P<0.05).Compared with blank control group, the number of inflammatory cells(mainly lymphocytes and monocytes) in the subarachnoid space of the rats in model group was significantly increased(P<0.01);compared with models group, the number of inflammatory cells in the subarachnoid space in different doses of Zhengqingfengtongning groups and sumatriptan group were significantly reduced(P<0.01). Conclusion Zhengqingfengtongning may inhibit the ANI of the rat model with migraine, suggesting that Zhengqingfengtongning has the effect of anti-inflammatory and analgesia in treatment of migraine.

Objective To construct the RNAi plasmid targeting P27RF-Rho gene, and to explore the effect of P27RF-Rho gene silencing on the proliferation of hepatocelluar carcinoma cells Bel7402. Methods The synthetic oligonucleotide fragment was cloned into RNAi plasmid, and the constructed recombinant plasmid was transfected into the hepatocelluar carcinoma Bel7402 cells for P27RF-Rho gene silencing.This experiment was divided into Bel7402 group, Scramble-siRNA group and P27RF-Rho-siRNA group.The transfection of Bel7402 cells was observed by fluorescence microscope.The gene silencing effect was detected by RT-PCR, and the proliferation of transfected hepatocelluar carcinoma Bel7402 cells was determined by drawing growth curves, and the ability of clone formation was measured by Colony formation assay.The expression levels of P16, CyclinE, MMP-9 and CDK-5 mRNA were detected by RT-PCR. Results The expression level of P27RF-Rho gene in the Bel7402 cells in P27RF-Rho-siRNA group was obviously lower than those in Bel7402 group and Scramble-siRNA group(P<0.01).The growth speed of Bel7402 cells in P27RF-Rho-siRNA group was lower than those in Bel7402 group and Scramble-siRNA group (P<0.05).Compared with Bel7402 group and Scramble-siRNA group, the expression levels of CyclinE, MMP-9 and CDK-5 mRNA in P27RF-Rho-siRNA group were notably decreased (P<0.01), whereas the P16 mRNA expression level was significantly increased (P<0.01). Conclusion The proliferation of hepatocellular carcinoma Bel7402 cells could be controlled by inhibiting the expression of P27RF-Rho gene.

Objective To express the mycobacteriophage D29 lysin gp10 in prokaryotic cells, and to investigate its antibacterial activities of alone and combined with isoniazid(INH)to Mycobacterium tuberculosis(MTB) in vitro. Methods The gp10 gene was amplified by PCR using the genome of mycobacteriophage D29 as a template and cloned into pET32a(+) vector. The constructed vector pET32a(+)-gp10 was identified by restriction enzyme digestion and nucleotide sequencing, and transformed to E.coli BL21(DE3) for expression. The minimal inhibitory concentration(MIC) of recombinant protein gp10 was detected by resazurin drugs combination microtiter assay (REDCA) for MTB standard strain H₃₇Rv and clinical isolate of multidrug-resistant MTB, the fractional inhibitory concentration index(FICI) was used to the antibacterial activity between INH and gp10 in MTB H₃₇Rv. Results The gp10 at a length of 1 479 bp was amplified by PCR, the recombinant plasmid pET32a(+)-gp10 was digested by EcoRⅠ and NotⅠ for 5 900 and 1 479 bp bands, gp10 sequence analysis was consistent with the sequence in GenBank, and it expressed a target protein with a relative molecular mass about 75 200. The MICs of recombinant protein gp10 were 256 mg·L^-1 against standard strain H₃₇Rv and clinical isolate of multidrug-resistant MTB. The FICI of combined INH and gp10 was 0.5 for MTB H₃₇Rv. Conclusion The recombinant protein gp10 is expressed successfully, and it shows good antibacterial activity against MTB H₃₇Rv and clinical isolate of multidrug-resistant MTB. Synergism in MTB H₃₇Rv is observed with gp10 combined with INH in vitro.

Objective To observe the lipid peroxidation of dibutylphthalate(DBP) on the reproductive system of the male mice and its influence in the antioxidant enzyme activities, and to investigate the possible mechanism of DBP to the reproductive system of the male mice. Methods 40 healthy male ICR mice were randomly divided into control group, 200 mg·kg^-1 DBP group, 400 mg·kg^-1 DBP group, and 800 mg·kg^-1 DBP group(n=10).The mice in DBP groups were administrated with 200, 400 and 800 mg·kg^-1·d^-1 DBP by gavage.The mice in control group were only received corn solvent.5 mice in each group were sacrificed respectively 2 and 4 weeks later, and their testis were collected.The malondialdehyde (MDA) levels, activities of glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) in testis tissue of the mice in various groups were detected. Results Two weeks after exposure, the MDA levels in testis tissue of the mice in 400 and 800 mg·kg^-1 DBP groups were significantly higher than those in control group and 200 mg·kg^-1 DBP group(P<0.05);the SOD activities in 400 and 800 mg·kg^-1 DBP groups were lower than that in 200 mg·kg^-1 DBP group (P<0.05).Four weeks after exposure, the testis organ coefficients in 400 and 800 mg·kg^-1 DBP groups were lower than those in control group and 200 mg·kg^-1DBP group (P<0.05);the MDA levels in 400 and 800 mg·kg^-1 DBP groups were higher than that in control group (P<0.05), and the MDA level in 200 mg·kg^-1 DBP group was lower than that in control group (P<0.05).The SOD activity in testis tissue of the mice in 800 mg·kg^-1DBP group was significantly lower than those in control group, 200 mg·kg^-1 DBP group and 400 mg·kg^-1 DBP group (P<0.05);the GSH-Px activities in testis tissue of the mice in DBP groups were significantly higher than that in control group (P<0.05). Conclusion DBP can induce the lipid peroxidation of the mouse testis and induce oxidative stress, and lead to the descending of testicular antioxidant capacity, then Results in reproductive dysfunction.

Objective To detect the influence of hesperetin in the growth of tongue cancer cells and the activation of Notch1 signaling and to explore its mechanism. Methods The Tca8113 cells were cultured and divided into DMSO control and 25, 50, 75, 100, 125 μmol·L^-1 hesperetin groups.Then MTT assay was performed to detect the inhibitory rate of proliferation of Tca8113 cells after treatment with different concentrations of hesperetin.Flow cytometry was used to detect the the percentage of apoptotic cells and the distribution of cell cycle, and qPCR and Western blotting Methods were used respectively to analyze the relative expression levels of Notch1 and Hes-1 mRNA and protein. Results The MTT Results revealed that compared with DMSO control group, the inhibitory rates of proliferation of Tca8113 cells in 125 μmol·L^-1 hesperetin groups at 24, 48, and 72 h(7.88%±1.9%, 26.28%±2.2%, 47.05%±1.9%)were significantly increased(P<0.05).Moreover, after hesperetin treatment for 72 h, the inhibitory rates of proliferation of the Tca8113 cells in 25, 50, 75, 100, and 125 μmol·L^-1 hesperetin groups were (6.14±1.2)%, (28.69±2.1)%, (28.33±2.6)%, (45.26±3.0)%, and (47.05±1.9)%, respectively; hesperetin inhibited the growth of Tca8113 cells in a time- and dose-dependent manner.The flow cytometry Results showed that after hesperetin treatment for 72 h, compared with DMSO control group, the apoptotic rates of the Tca8113 cells in 50 and 100 μmol·L^-1 hesperetin groups were increased significantly from (2.9±0.5)% to (23.4±1.7)% and (35.1±1.9)%(P<0.05);the percentages of the Tca8113 cells in G₀/G₁ phage in 50 and 100 μmol·L^-1 hesperetin groups were increased significantly from (18.6±1.3)% to (33.4±1.5)% and (40.5±1.9)% (P<0.05), and the percentages of the Tca8113 cells in S phage were decreased from (70.3±2.5)% to (56.8±2.0)% and (48.7±1.8)%(P<0.05).The qPCR and Western blotting Results showed that the expression levels of Notch1 and Hes-1 mRNA and protein were all increased compared with DMSO control group(P<0.05). Conclusion Hesperetin can inhibit the growth and induce the apoptosis and G₀/G₁ arrest in the Tca8113 cells, and its mechanism may be related to activating the Notch1 signaling.

Objective To observe the influence of aerobic exercise in the renin-angiotensin system (RAS) and cardiac function in the obese hypertension (OH) rats, and to clarify the protective effect of aerobic exercise on OH. Methods The male healthy Sprague-Dawley rats were used to induce rat model of OH by high fat diet.Thirty successfully established OH rats were randomly assigned into sedentary group (OH+S) and exercise group (OH+E) with 15 rats for each group.Meanwhile, 30 rats fed with regular diet were randomly assigned into sedentary group (N+S) and exercise group (N+E) with 15 rats for each group.The rats in OH+E group and N+E group underwent treadmill running at 18 m·min^-1 (60 min each, 5 times a week).Eight weeks later, the systolic blood pressure (SBP), body weight, serum levels of renin, angiotensin Ⅱ and aldosterone of the rats in various groups were measured.Moreover, the obesity related indexes, including triacylglycerol (TG), total cholesterol (TC), Lee's index and fat coefficient (FC), and fat weight were recorded.The PowerLab data system was employed to record the cardiac function, such as left ventricular end diastolic volume (EDV), left ventricular end systolic volume (ESV), fractional shortening (FS) and ejection fraction (EF). Results Compared with OH+S group, the SBP, body weight, serum levels of renin, angiotensin Ⅱ and aldosterone, obesity related indexes and fat content of the rats in OH+E group after exercise were decreased (P<0.05).The FS and EF of the rats in OH+E group were increased and the EDV and ESV were decrased compared with OH+S group (P<0.05).However, the above indexes of the rats inOH+E group were still different from N+S group and N+E group (P<0.05). Conclusion Aerobic exercise can attenuate the RAS activation and improve cardiac function, also improve the symptoms of hypertension and obesity.

Objective To investigate the effect of Wutou Injection on a variety of different stages of inflammation models, and to initially clarify its anti-inflammatory mechanism. Methods The Wistar rats were randomly divided into model group, low, medium, and high doses (0.4, 0.8, 1.6 mg·kg^-1) of Wutou Injection groups, and positive control group (etanercept, 10 mg·kg^-1);the rats in model group and positive control group at the 1st and 4th day were administered subcutaneously;the rats in Wutou injection groups were intramuscularly administered for 7 consecutive days.The ICR mice were randomly divided into blank control group, low, medium and high doses (0.5, 1.0, 2.0 mg·kg^-1) of Wutou Injection groups, and positive control group (etanercept, 14 mg·kg^-1);the mice in model group and positive control group at the 1st and 4th day were administered subcutaneously;the mice in Wutou Injection groups were intramuscularly administered for 7 consecutive days.The acute and chronic animal models, such as carrageenan-induced paw oedema in the rats, formaldehyde-induced paw oedema in the rats, CMC-Na induced air pouch leukocyte migration in the rats, granuloma induced by cotton ball in the rats, the permeability of blood capillary in abdominal cavity of the mice, were selected to evaluate the paw swelling rates, the number of leukocytes from subcutaneous CMC-Na cysts, organ coefficient, and permeabilities of blood capillary in abdominal cavity. Results Compared with model control group, the paw swelling rates of the rats in medium and high doses of Wutou Injection groups were decreased 1, 4, 5 and 6 h after inflammation induced by carrageenan (P<0.05 or P<0.01); the paw swelling rates of the rats in medium and high doses of Wutou Injection groups were decreased from 6 to 48 h after inflammation induced by formaldehyde (P<0.05 or P<0.01);the number of leukocytes from subcutaneous CMC-Na cysts of the rats in high dose of Wutou Injection group was decreased (P<0.05); the coefficient of granuloma induced by cotton ball of the rats in low, medium and high doses of Wutou Injection groups were decreased (P<0.05 or P<0.01);and the organ indexes had no changes(P>0.05).Compared with blank control group, the permeabilities of blood capillary in abdominal cavity of the mice in low, medium, and high doses of Wutou Injection groups had no significant changes (P>0.05). Conclusion Wutou Injection has anti-inflammatory effects on the different stages of inflammation animal models.

Objective To investigate the different effects of 5-hydroxytryptamine-7(5-HT₇) on the electrical activities of 5-HT neurons in nucleus raphes dorsalis(DRN) of the normal and parkinson's disease(PD) rats, and to clarify the properties of 5-HT₇ receptor and the changes in the PD rats. Methods 28 male SD rats were randomly divided into sham opration group (n=12) and PD group (n=16).6-OHDA was injected in the subtantia nigra pars compacta(SNc)of the rats in PD group, and the same dose of normal saline was injected in the SNc of the rats in sham opration group.The rats in two groups were injected with different doses of (40-640 μg·kg^-1, i.v.) 5-HT₇ receptor agonist AS19, and the changes of firing rates in 5-HT neurons of the rats were observed by extracellular electrophysiological recording in DRN.5-HT₇ receptor antagonist SB269970 was injected to observe the sensitivities to agonist and the antagonist of the rats in PD group, and compared with sham operation group. Results Systemic administration of the selective 5-HT₇ receptor agonist AS19(40-640 μg·kg^-1, i.v.)increased the firing rate of 5-HT neurons of the rats in sham operation group, the mean firing rate of 5-HT neurons in the DRN was changed to 2.35±0.16(P<0.001), and completely reversed by the subsequent administration of SB269970(200 μg·kg^-1, i.v.), the mean firing rate of 5-HT neurons in the DRN was changed to 0.37±0.03.The same dose of AS19 had no affection in the rats in PD group(P=0.218). Conclusion The regulation effect of 5-HT₇ receptor on the 5-HT neurons in DRN of the PD rats is decreased.

Objective To study the effects of hyperbaric oxygen at different intervention time on the apoptosis and functional recovery following spinal cord injury(SCI) in the rats and to investigate the ideal application method of hyperbaric oxygen, and to provide some experimental basis for clinical hyperbaric oxygen therapy for SCI. Methods 160 SD rats were randomly divided into normal injury group, hyperbaric oxygen preconditioning(HBO-PC) group, hyperbaric oxygen therapy(HBOT)group and HBO-PC+HBOT group.The rat models of SCI were established with Allen's dropping weight technique.The rats in normal injury group weren't treated with any intervention;the rats in HBO-PC group were treated with hyperbaric oxygen intervention 10 d before modeling;the rats in HBOT group were treated with hyperbaric oxygen intervention only after modeling;the rats HBO-PC+HBOT group were treated with hyperbaric oxygen intervention before and after modeling.The segments of injured spinal cord were collected from the rats in 4 groups on the 1st, 2nd, 7th and 14th day after injury.The expression levels of Fas and FasL were detected with immunnohistochemistry PV-9000 two steps method.The apoptotic index(AI) was detected with TUNEL technique.The neurological function of spinal cord was assessed by BBB score. Results Compared with normal injury group, the expression levels of Fas and FasL of the rats in HBO-PC group, HBOT group and HBO-PC+HBOTgroup were decreased significantly(P<0.01);there were significant differences between HBO-PC+HBOT group and HBO-PC group, HBOT group(P<0.05), and there was no significant difference between HBO-PC group and HBOT group(P>0.05).Compared with normal injury group, the AI of the cells in HBO-PC group, HBOT group and HBO-PC+HBOT group were decreased significantly(P<0.01);compared with HBO-PC group and HBOT group, the AI of the cells in HBO-PC+HBOT group was decreased significantly(P<0.05);there was no significant difference between HBO-PC group and HBOT group(P>0.05).Compared with normal injury group, the BBB scores of the rats in HBO-PC group, HBOT group and HBO-PC+HBOT group 7 and 14 d after operation were increased significantly(P<0.01);compared with HBO-PC group and HBOT group, the BBB scores in HBO-PC+HBOT group were increased significantly (P<0.05 or P<0.01);compared with HBO-PC group, the BBB scores in HBOT group were increased significantly (P<0.05). Conclusion Hyperbaric oxygen can inhibit the apoptosis after SCI, which contributes to the recovery of locomotor performances in the rats.The best intervention time is in front of the damage.The method of HBO-PC combined with HBOT is the best way for the treatment of SCI.

Objective To investigate the effect of Raman spectroscopy for isoprenaline hydrochloride(Iso)-induced liver injury after myocardial fibrosis of ischemia in the rats, and to provide the basis for the application of Raman spectroscopy. Methods 20 Wistar rats were randomly divided into control group, Iso-4h group, Iso-24h group, and Iso-1 week group (n= 5).Except the normal group, the rats in other groups were subcutaneously injected with Iso.The pathological changes of heart and liver tissues were analyzed by HE staining.The absorption spectra of proteins in the liver tissue was analyzed by UV and Raman spectroscopy. Results The pathological observation Results showed that the cardiomyocytes of the rats in control group arranged in neat rows with clear stripes, obvious nucleus and no cell swelling;the cardiomyocytes of the rats in Iso-24h group appeared the more necrosis limting under endocardium;the myocardium of the rats in Iso-1 week group appeared the multiple scattered necrosis with fibroblasts and collagen fiber.The hepatic lobules of the rats in control group had clear boundaries without necrosis;the hepatic lobules of the rats in Iso groups had focal necrosis, and the liver cells appeared watery degeneration;with the prolongation of the Iso injection time, the degree of liver lesion was serious.The UV spectroscopy showed that the myocardial protein spectrum of the rats in Iso-24h group moved 1 unit to the left or the right, and the spectrum in Iso-1 week group became nromal compared with control group;the liver protein spectrum of the rats in Iso-24h group moved 2 units, and the spectrum in Iso-1 week gradually stabilized compared with control group.The Raman spectrocopy showed that the absorption peak of liver tissue in Iso-24h group appeared at 1 590 nm, but it wasn't found in Iso-1 week group. Conclusion Liver damage is found when Iso induces myocardium injury, and Raman spectroscopy is a more sensitive detection method for liver damage.

Objective To investigate the influence of TXNIP in the biological characteristics of gastric cancer MKN-45 cells, and to clarify the relationships between TXNIP and the carcinogenesis and progression of gastric cancer. Methods The cDNA of TXNIP was subcloned into a constitutive vector pcDNA3.1 followed by transfection in MKN-45 cells by using liposome.RT-PCR and immunocytochemistry were used to detect the expressions of TXNIP gene and protein in MKN-TXNIP cells.Each detection experiment was divided into MKN-TXNIP group, MKN-PC group and MKN-45 group (blank control group).Flow cytometry was used to analyze the apoptosis and cell cycles of gastric carcinoma cells.The cell growth curves and the colony assay were used to detect the growth and proliferation of MKN-45 cells.Transwell chamber was used to detect the invasion. Results Compared with MKN-PC group and MKN-45 group, TXNIP gene could express stably in MKN-TXNIP group.The growth speed of the MKN-45 cells in MKN-TXNIP group was slower than those in MKN-45 group and MKN-PC group(P<0.05).The Results of colony formation assay showed that the colony formation rate in MKN- TXNIP group was lower than those in MKN-45 group and MKN-PC group (P<0.05).The percentage of the cells in G₀/G₁ phase in MKN-TXNIP group was higher significantly(P<0.05), and the percentage of the cells in G₂/M plase was slower than those in MKN-45 group and MKN-PC group.The flow cytometry Results showed that the apoptotic rates of the cells in each group were about 2.0%, and there were no significant differences between various groups(P>0.05).The Results of migration assay suggested that the cell migration rate of the MKN-45 cells in MKN-TXNIP group was significantly lower than those in MKN-45 group and MKN-PC group (P<0.05). Conclusion TXNIP gene can inhibit the growth, proliferation, division and invasion of gastric cancer cells.It can also influence the cell cycle, but it only has little influence in the apoptosis of MKN-45 cells.

Objective To investigate the distribution of common pathogens in different departments and drug resistance of these pathogens in the patients with urinary tract infections in our hospital, and to provide reference basis for selecting reasonable, timely and personal treatment plan with effective antibiotic agents for the patients in different clinical departments. Methods 252 urine samples with positive culture from the hospitalized patients were collected and the sensitivity Results and bacterial resistance rates were obtained.The distribution of 270 strains isolated from 252 samples and the susceptibility Results were analyzed and the drug resistance of bacteria in different departments was compared. Results In 270 strains, the number of gram-negative bacteria was 202 (74.81%), Escherichia coli was the most common pathogen (121, 44.81%), followed by Klebsiella pneumoniae (24, 8.89%), Pseudomonas aeruginosa (10, 3.70%), Klebsiella oxytoca (9, 3.33%);In gram-positive bacteria, feces Enterococcus was the most common pathogen (20, 7.41%), followed by Viridans (12, 4.44%), Enterococcus faecalis (10, 3.70%);ESBLs-producing pathogens were 28 strains which accounted for 13.86% of all bacteria.The number of meropenem-resistant bacteria was 8 strains.All feces Enterococci were sensitive to linezolid and teicoplanin, but of which the resistance rate was 5.0% for vancomycin.The pathogens were different in different departments, such as the number of feces Enterococcus in ICU was the most, followed by Escherichia coli;in VIP ward, the infection rates of Enterococci and Escherichia coli in the patients were the same;in other major departments the Escherichia coli had the highest proportion of bacteria. Conclusion Bacilus is the main pathogenic bacterium in the patients with urinary tract infections, but the coccus should be paid attention.The pathogenic bacteria have resistance to many drugs, and the main pathogenic bacteria vary in different departments, so the personal treatment plan for different patients should be made.

Objective To explore the changes in the number of peripheral blood cells in the patients with chronic hepatitis C(CHC) after treated with interferon alpha 2b(IFN-α2b) combined with ribavirin, and to clarify the impact factors of the number of peripheral blood cells. Methods A total of 343 CHC patients with symptoms of chronic hepatitis and positive anti -HCV and HCV RNA were selected.All patients received IFN-α2b (5 000 000 U, every other day, subcutaneous)combined with ribavirin (15 mg·kg^-1·d^-1, oral)for 48 weeks.The number of peripheral blood cells and serum HCV RNA level were assessed at 12, 24, 36, 48, 60, 72, and 96 weeks.The patients who developed neutropenia, anemia and thrombocytopenia would be treated according to "2010 Hepatitis C Treatment Guidelines". Results The prevalence of neutropenia, anemia and thrombocytopenia were 40.5%(139), 48.4%(166) and 39.9%(137), respectively.There were changes in the number of peripheral blood cells of individual patients after treatment.The most of changes occured from the beginning to 12 weeks.The number of neutrophils, hemoglobin and plateletsin were significantly decreased in 2 weeks, while the minimum value appeared at the 12th weeks followed by a slow rise.At the end of treatment (48 weeks), although the recovery was obvious, but not yet reached the pre-treatment levels.They recoved to the pre-treatment levels at 60 to 72 weeks.The baseline of absolute neutrophil count(ANC), plateletsin(PLT), aspartate transaminase(AST), alanine transaminase(ALT) and liver fibrosis were associated with neutropenia (P<0.05).The gender and baseline of hemoglobin levels were related to anemia(P<0.05).Gender, baseline of ANC, PLT, AST, ALT, γ-glutamyl transferase (γ-GT) and liver fibrosis were associated with thrombocytopenia(P<0.05). Conclusion The number of peripheral blood cells is significantly decreased in 2 weeks after anti-virus therapy, and reaches a minimum at 12 weeks, which reminds that clinicians should perform intervention and follow up timely to avoid serious adverse events.The occurrence and severity of reduction in the number of peripheral blood cells could be assessed according to the baseline gender, level of peripheral blood cells, liver function and liver fibrosis.

Objective To compare the efficacy between cannulated screw band(CSB) and Kirschner tension band(KTB) in the treatment of patella fracture with Meta-analysis, and to provide an objective evidence for clinical selection of operation Methods in the treatment of different types of patella fracture. Methods The Pubmed databases, the Cochrane Central Register of Controlled Trials, EMbase, CNKI databases, CBM databases and Wanfang databases(1997, 01-2014, 09), were retrievaled by two reseachers, respectively; several related journals were searched by hand.The control trials comparing CSB with KTB in the adults were collected.Two researchers independently assessed the trial quality and extracted the data into an electrical sheet.The data of the treatment outcome, the time of fracture healing, the incidence of complications and the operation time from these studies were abstracted and synthesized by Meta-analysis with RevMan 5.1 software. Results A total of 10 studies involving 734 patients were included.The Results of Meta-analysis showed that in CSB group the treatment outcome was increased(OR=4.00, 95%CI:1.56-10.27, P=0.004), the time of the fracture healing was shortened(MD=-0.76, 95%CI:-1.03--0.48, P<0.01), the incidence of complications was decreased(OR=0.07, 95%CI:0.03-0.17, P<0.01)and the operation time was shortened(MD=-3.91, 95%CI:-7.68--0.13, P=0.04)compared with KTB group. Conclusion CSB is superior to KTB in the intraoperative treatment outcome, time of fracture healing, incidence of complications, and operation time.CSB is an ideal operation to treat the patients with patella fracture.

Objective To investigate the curative effect of dextrose used in knee osteoarthritis (KOA) by applying a Meta-analysis, and to provide evidence for dextrose prolotherapy of KOA. Methods The literatures about the effect of dextrose prolotherapy in the treatment of KOA were retrieved from Pubmed/Medline, Scopus the and Cochrane Library.All the related literatures were checked and qualified in the method of evidence-based medicine.The improvement of pain, physical function, and stiffness relief were analyzed by RevMan 5.1 between dextrose group and control group. Results Five case-control studies with a total of 355 knees from 234 patients were included.Meta-analysis showed that dextrose prolotherapy had both short-term and long-term effects on the pain relief(short term:SMD =-1.45, 95% CI:-2.42--0.48, P=0.003;long-term:SMD =-2.20, 95% CI:-3.45--0.94, P=0.000 6) in addition to physical function(SMD =-2.00, 95% CI:-2.84--1.55, P<0.000 01). The stiffness improvement was not significant(SMD =-0.05, 95% CI:-1.02-0.03, P=0.07). Conclusion For KOA, the dextrose prolotherapy could improve the symptoms of pain and physical function.But further studies are needed to exam its effects on stiffness.

Objective To explore the association between HLA-DRB1 gene rs2308765, rs9269186 and rs3135388 polymorphisms and essential hypertension(EH), and to clarify the association between single nucleotide polymorphisms(SNPs) of HLA-DRB1 gene and EH. Methods Using case-control study design, 161 patients with EH were recruited in case group and 147 healthy persons were selected as control group.The related factors of EH were collected and the polymorphisms of HLA-DRB1 rs2308765, rs9269186 and rs3135388 sites were detected with PCR-LDR. Results The body mass index(BMI), waist circuit(WC), blood glucose(BG), total cholesterol(TC), triglyceride(TG) and low density lipoprotein cholsterol (LDL-C) levels of the patents in case group were significantly higher than those in control group(P<0.05 or P<0.01), while the level of high density lipoprotein cholesterol (HDL-C) was significantly lower than that in control group(P<0.01).After the potential confounders in the model of Logistic regress were adjusted, the high BMI (OR=1.140, 95%CI:1.040-1.249)and TG (OR=1.215, 95%CI:1.016-1.452) were detected as the independent risk factors of EH.The frequency of the genotype GG and the frequency of G allele of rs2308765 site in case group were significantly lower than those in control group(χ²=4.666, P=0.031;χ²=4.485, P=0.034).The distribution of genotypic and allelic frequencies of rs3135388 and rs9269186 sites were not significantly different between case group and control group(P>0.05).Among the four environmental factors, such as BMI, TG, TC and HDL-C, only rs2308765 polymorphisms had interaction with TG(P<0.05). Conclusion The BMI, TG and SNPs of rs2308765 site of HLA-DRB1 gene are associated with EH.High BMI and high TG are the risk factors of EH, and rs2308765 G/T mutation might be the risk factor of EH.

Objective To investigate the expression levels of serum microRNA-181a and microRNA-20a in the patients with multiple myeloma (MM), and to explore the clinical significances of microRNA-181a and microRNA-20a in the occurrence and development of MM. Methods 32 patients with M and 12 patients with other blood diseases, from Department of Hematology of First Affiliated Hospital, School of Medical Sciences, Xi'an Jiaotong University, during 2013.01-2014.05, were chosen.All the subjects accorded with the diagnostic criteria from Blood Disease Diagnosis and Curative Effect of Standard, excluding anybody who was in a stable condition with other organ failure such as heart, lung, and liver.And at the same time, 20 healthy subjects were selected as control group.The expression levels of microRNA-181a and microRNA-20a in serum of the patients with MM, the patients with other disease and healthy individuals were examined by Real-time polymerase chain reaction (Real-time PCR).The serum β2 microglobulin (β2- MG) levels, lactate dehydrogenase (LDH) activities, albumin (ALB) levels, free light chain (FLC) levels and M protein percentages were detected at the same time in three groups, and then the differences were compared. Results The expression levels of serum microRNA-181a and microRNA-20a in three groups were 0.0452 and 5.879, 0.000 and 0.072, 0.004 and 1.159, respectively;the differences showed statistically signiifcance(H=15.218, 9.891;P=0.000, 0.007).The differences between MM group and normal control group were statistically significant (Z=-2.702, -1.979;P=0.005, 0.048) and the differences between MM group and other blood diseases group were also statistically significant (Z=-3.163, -2.722;P=0.001, 0.005).The expression levels of microRNA-181a and microRNA-20a were positively correlated with the M protein percentage(r=0.574, 0.739;P=0.032, 0.003);the expression level of microRNA-181a was positively related with the serum β2-MG and serum free FLC levels(r=0.552, 0.780;P=0.041, 0.001). Conclusion The microRNA-181a and microRNA-20a highly express in the serum of the patients with MM, which suggests that microRNA-181a and microRNA-20a might play a role in the development of MM and they may become the diagnosis markers of MM;microRNA-181a is expected to be an index to predict the poor prognosis of MM.

Objective To analyze the risk factors associated with the recurrence in the patients with hepatocellular carcinoma (HCC), and to evaluate the value of serum prealbumin (PA) in predicting the recurrence in the patients with HCC after operation. Methods A retrospective study of the 143 patients, diagnosed by pathology with HCC after hepatectomy (solitary, tumor size≤5 cm and no metastases or major vascular invasion), was performed and the patients were divided into non-complication group and complication group by the occurrence of postoperative complication.The differences that related to tumor recurrence in two groups were evaluated using Kaplan-Meier curves. Results The patients with postoperative complications showed a higher descend rang of PA (P<0.05).The overall median disease free survival(DFS) and the rate of recurrence of 143 patients were 43 months and 44.7%, respectively. The 1-, 2-, 3- year recurrence rates of the patients after hepatectomy were 29%, 39% and 49%, respectively.The postoperative DFS rates of 1-, 2-, 3- year were 67%, 52%, and 47%, respectively, in the patients with HBV-DNA<10³IU·mL^-1, while they was 67%, 52%, and 47% in the patients with HBV-DNA≥10³IU·mL^-1.The postoperative DFS rate of 1-, 2-, 3- year were 91%, 81%, 64% respectively in the patients with PA decline <43%, while they were 66%, 55%, and 47% in the patients with decline PA≥43%. Conclusion The HBV-DNA level and the decline of postoperative PA are the risk factors of recurrence HCC of after hepatectomy.The recurrent time of the patients with HCC varies with the risk factors.

Objective To analyze the clinical pathologic characteristics, treatment and prognosis of 476 breast cancer patients, and to explore the related factors influencing the prognosis of breast cancer. Methods 476 breast cancer patients received postoperative adjuvant therapy were followed up who were diagnosed as the pathologic stage Ⅰ-Ⅲ from 2007 to 2010.The median follow-up time was 50.5 months.Kaplan-Meier method was used to draw the survival curve, and Log-rank test was used to compare the survival rates;COX proportional hazards model was used to analyze single factors and multiple factors.The risk factors related to the prognosis of breast cancer were analyzed. Results The 3-year disease free survival rate of the patients was 94.3%.The single factor analysis showed that age, tumor size, the number of metastatic lymph nodes, vascular tumor emboli, hormone receptor status, HER-2 expression and molecular subtypes were the influencing factors of disease free survival(DFS) (P<0.05).The smaller age, the larger neoplasm, the more metastatic lymph nodes, the more vascular tumor emboli, the lower positive rate of hormone receptor, the over-expression of HER-2, the molecular subtypes of HER-2 over-expression and the basal-like subtype had poor prognosis among them.The multifactor analysis demonstrated that hormone receptor status and HER-2 expression were independent factors influencing DFS (P<0.05).The 3-year overall rate of the patients was 97.6%.The single factor analysis showed that the tumor size, the number of metastatic lymph nodes, vascular tumor emboli, hormone receptor status and HER-2 expression were the influencing factors of overall survival (OS)(P<0.05).The multiple factor analysis demonstrated the number of metastatic lymph node, hormone receptor status and HER-2 expression were independent factors of OS (P<0.05). Conclusion The number of metastatic lymph nodes, hormone receptor status and HER-2 expression are significantly independent indicators of prognosis in the patients with breast cancer respectively.

Objective To discuss the efficacy and safety of intravitreal injection of ranibizumab combined with laser photocoagulation in the treatment of diabetic macular edema (DME), and to explain the advantages of this combination treatment method in treating DME. Methods 71 patients (85 eyes) with macular edema due to diabetic retinopathy(DR) were randomly divided into combination treatment group (42 eyes received intravitreal injections of ranibizumab combined with laser photocoagulation treatment) and photocoagulation group (43 eyes received laser photocoagulation treatment only).The changes of the best corrected visual acuity (BCVA), the fovea centralis thickness (CMT), and the intraocular pressure before and after treatment were observed and analyzed. Results The BCVA of the patients in combination treatment group was improved after treatment than before (P<0.05), but there was no difference of BCVA in photocoagulation group before and after treatment(P>0.05).The BCVA improvement in combination treatment group was statistically more significant (P<0.05) compared with photocoagulation group in 1 month, 3 months and 6 months after laser photocoagulation treatment.Compared with before treatment, the CMT of the patients in two groups in 1 month, 3 months, 6 months after laser photocoagulation treatment were reduced.At each stage, the CMT of the patients in combination treatment group were lower than those in photocoagulation group (P<0.05).No complications of glaucoma, cataract, vitreous hemorrhage, retinal detachment, choroidal detachment, endophthalmitis and no systemic complications were observed in all patients. Conclusion For the patients with DME, the combination treatment with intravitreal injection of ranibizumab and laser photocoagulation has a more quickly and better therapeutic effect than the sole laser photocoagulation treatment in reducing the macular edema and improving the BCVA, also has less complication.

Objective To study the relationship between ovulation induction treatment and endometrial polyps formation in the patients with infertility, and to clarify the factors which influence the formation of endometrial polyps during ovulation induction treatment. Methods The data was collected from 76 infertile patients who had received ovulation induction treatment and came to outpatient service for hysteroscopy.The statistical analysis of the relationship between ovulation induction and endometrial polyps formation was performed in terms of infertility types, infertility duration, ovulation induction cycles and presence of gonadotrophin releasing hormone analogue(GnRHa) in the treatment. Results The occurence rates of endometrial polyps between the patients with different infertility types (χ²=0.071, P=0.790) or the different durations of intertility (χ²=1.561, P=0.458)had no significant differences while the occurrence rates between the patients with different ovulation cycles (χ²=4.992, P=0.025) or whether to use GnRHa (χ²=18.899, P=0.000) had significant differences.The occurrence rate of endometrial polyps was increased as the cycles of ovulation induction was increased.The occurrence rate of endometrial polyps was twice higher in the patients with three or more cycles of ovulation induction than that in the patients with one or two cycles (52.4% vs 25.5%, P=0.025).Meanwhile, the occurrence rate in the patients treated with GnRHa was significantly lower than that in the patients treated without GnRHa (16.0% vs 65.4%, P=0.000). Conclusion The increase of cycles of ovulation induction is a risk factor for endometrial polyps while the presence of GnRHa in the treatment is a protective factor against the formation of endometrial polyps.

Objective To perform the exercise test of low-frequency repetitive nerve stimulation(RNS) in the patients with myasthenia gravis(MG), and to clarify the diagnostic value of exercise test in the patients with MG. Methods 30 patients with MG were collected.The RNS at rest and exercise test in facial nerve were performed in all patients.Exercise test was performed at fatigue 1 min and 2 min, then the RNS was performed at the ending of fatigue, 1 min and 2 min after fatigue, respectively.The decreasing degree of compound muslce action potential (CMAP) amplitude at different time points were analyzed. Results The CMAP amplitudes in exercise test of RNS were decreased significantly when compared with RNS at rest at different time points, especially at fatigue 2 min and rest 1 min.Amang 30 patients, 12 patients had normal RNS at rest, but all of them had abnormal RNS after exercise test. Conclusion Exercise test of RNS has the diagnostic value in the patients with MG, especially in the patients with normal RNS at rest.Fatigue 2 min and rest 1 min could be considered when performing exercise test of RNS.

Objective To construct the pGEX-4T-1/hZimp10 recombinant plasmid and to express in E.coli, and to purify the GST-hZimp10 fusion protein to prepare anti-hZimp10 antibody. Methods The corresponding DNA fragment with the N-terminal 128 amino acids of hZimp10 protein was amplified by PCR using the template plasmid pcCDNA3.1-Flag-hZimp10 and then cloned into the expression vector pGEX-4T-1.After identified by enzyme digestion, the recombinant clone was transformed into the expression cells of E.coli BL21.The GST-hZimp10 fusion protein was induced by IPTG and then purified as an antigen into the rabbits to prepare the polyclonal antibody.At last, the titer and specificity of the antibody were analyzed with indirect ELISA and Western blotting method. Results The pGEX-4T-1/hZimp10 recombinant plasmid was constructed successfully.It contained a 384 bp insert fragment identified by BamHⅠ and XhoⅠ double digestion, and the result was consistent with expectations.The SDS-PAGE electrophoresis showed the fusion protein was well expressed and protein molecular weight was consistent with that expected.The indirect ELISA and Western blotting Results indicated that anti-hZimp10 antibody showed high titer (1:100 000) and high spencificity in the LNCaP cells. Conclusion GST-hZimp10 fusion protein is obtained successfully and the anti-hZimp10 antibody is prepared.