Objective To investigate the roles of schisandrin B (SchB) on the apoptosis and invasion of human colon cancer SW480 cells and its influence in p38MAPK signaling pathway,and to clamify the mechanisms. Methods The SW480 cells were divided into control group,low dose of SchB group (20 μmol·L^-1,SchB₁ group),middle dose of SchB group (40 μmol·L^-1,SchB₂ group) and high dose of SchB group (80 μmol·L^-1,SchB₃ group).After the SW480 cells were treated with different doses of SchB,the inhibitory rates of proliferation of the SW480 cells were evaluated using MTT assay,the apoptotic rates were analyzed by flow cytometry using Annexin V-FITC and PI double staining methods,the invasion activities of SW480 cells were tested using Transwell chamber,and the expression levels of p-p38,p38,p-p53 and p53 were measured by Western blotting method. Results Compared with control group,the inhibitory rates of proliferation and the apoptotic rates of colon cancer SW480 cells in SchB₁,SchB₂ and SchB₃ groups were increased(P<0.01),the invasion rates and the number of SW480 cells penetrating the membrane were decreased (P<0.01), and the expression levels of p-p38 and p-p53 proteins were increased (P<0.05 or P<0.01). Conclusion SchB can inhibit the proliferation and invasion of human colon cancer SW480 cells and promote the apoptosis.p38MAPK signaling pathway may be involved in the inhibitory effect of SchB on the tumor cells.

Objective To investigate the protective effect of rhein lysinate (RHL) on the kidney of aging mice induced by D-galactose (D-gal) and to clarfy its mechanism,and to provide the basis for research on prevention of RHL on aging. Methods The models of aging mice were established by intraperitoneally injecting with D-gal.The mice were randomly divided into control,aging model,low and high doses of RHL groups.In addition to the control group,the mice in the other groups were injected subcutaneously with D-gal daily for 8 weeks,while the mice in control group were injected subcutaneously with the same amount of saline daily,and the mice in low and high doses of RHL groups were given 25 and 50 mg·kg^-1 RHL daily by gavage.The contents of malonaldehyde (MDA) and the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in serum and kidney tissue were detected by microplate reader according to the instruction of respective kits.The pathological changes of kidney tissue were observed by hematoxylin and eosin (HE) staining;the expression levels of age-related proteins were detected by Western blotting method. Results Compared with control group,the kidney index of the mice in aging model group was decreased (P<0.05);the activities of SOD and GSH-Px in serum and kidney tissue were decreased (P<0.05),while the content of MDA was increased (P<0.05).Compared with aging model group,the kidney indexes of the mice in 25 and 50 mg·kg^-1RHL groups were increased (P<0.05);the activities of SOD and GSH-Px in serum and kidney tissue of the mice were increased (P<0.05) and the contents of MDA were decreased (P<0.05).Compared with aging model group,the edema of renal tubular epithelial cells in kidney tissue was decreased in a dose-dependent manner in 25 and 50 mg·kg^-1 RHL groups.Compared with aging model group,the expression levels of SIRT1 in kidney tissue of the mice in 25 and 50 mg·kg^-1 RHL groups were increased (P<0.05),while the expression levels of p16 and p21 were decreased (P<0.05). Conclusion RHL can inhibit the oxidative stress and the edema of renal tubular epithelial cells and regulate the expressions of age-related genes to protect the kidney of aging mice.

Objective To investigate the possible mechanisms of sericin in treating diabetes mellitus by observing the effects of sericin on the expressions of tumor necrosis factor-α (TNF-α) and hepatocyte nuclear factor 4 alpha (HNF-4α) in liver tissue of the rats with type 2 diabetes mellitus. Methods 36 male SD rats were randomly divided into normal control group,diabetes model group and sericin treatment group (n=12).The type 2 diabetes mellitus rat models were induced by continuously intraperitoneal injection of streptozotocin(STZ).After successfully establishing the diabetes models,the rats in diabetes model group received any more treatments,and the rats in sericin treatment group were lavaged with sericin (2.4 g·kg^-1·d^-1) for 35 d. SP immunohistochemical staining,Western blotting and RT-PCR were used to detect the TNF-α and HNF-4α expressions in liver tissue of the rats. Results The TNF-α protein immune positive products were tan and brown granules and located in the cytoplasm of liver cells;the HNF-4α immune positive products were tan and located in the nucleus of liver cells. Compared with normal control group,the TNF-α and HNF-4α protein and mRNA expression levels in liver tissue of the rats in diabetes model group were increased obviously (P<0.01).Compared with diabetes model group,the TNF-α and HNF-4α protein and mRNA expression levels in liver tissue of the rats in sericin treatment group were decreased obviously (P<0.01). Conclusion Sericin may protect the liver injury in the rats with diabetes mellitus by down-regulating the TNF-α and HNF-4α expressions in liver tissue.

Objective To study the toxicity of near-infrared quantum dots (NIR QDs) linked to peptide containing the arginine-glycine-aspartic acid (RGD) sequence (NIR QDs-RGD) in the mice,and to clarify the biological safety of NIR QDs-RGD as a novel tumor treatment method. Methods The probe was prepared firstly by linking NIR QDs to the RGD peptide.Twenty-four BALB/C mice were randomly divided into four groups.In QD800-RGD one injection group,the mice were injected via tail vein with 100 μL QD800-RGD (equivalent to 200 pmol of QD800) on the first day;in PBS one injection group,the mice were injected similarly with 100 μL PBS;in QD800-RGD two injection group,the mice were repeatedly injected via tail vein with 100 μL QD800-RGD (equivalent to 200 pmol of QD800) on the first and seventh days;in PBS two injection group,the mice were repeatedly injected similarly with 100 μL PBS on the first and seventh days.Fourteen days after injection,toxicity tests were performed,including complete blood cells(white blood cells,red blood cells,hemoglobin,platelets,lymphocytes,and neutrophils) count and serum biochemical analysis (total protein,albumin,albumin/globulin,aspartate aminotransferase,alanine aminotransferase,and blood urea nitrogen).The coefficients of liver,spleen,kidney,and lung weight to body weight were calculated,and the activities of superoxide dismutase(SOD),glutathione(GSH),and malondialdehyde(MDA) contents in liver,spleen,kidney and lung tissues of the mice were detected.The histopathological examination was performed. Results Compared with PBS injcetion groups,the organ coefficients of the mice in QD800-RGD one injection group were decreased slightly,and the organ coefficients of the mice in QD800-RGD two injection group were increased,but the differences between four groups were not statistically significant (P>0.05).Compared with PBS injcetion groups,the number of leukocytes,erythrocytes,lymphocytes and the levels of hemoglobin and platelets were increased,and the number of neutrophils had no significant change;the number of red blood cells,lymphocytes and hemoglobin level in QD800-RGD two injection group were increased;the number of white blood cells and the level of platelets were decreased,and the number of neutrophils had no significant changes;but the differences were not statistically significant between four groups (P>0.05).Compared with PBS injection group,the total protein,albumin,albumin globulin ratio,and blood urea nitrogen in QD800-RGD one injection group were reduced;the aspartate aminotransferase and alanine aminotransferase were increased;the total protein and albumin were increased,the albumin globulin ratio,blood urea nitrogen,aspartate aminotransferase and alanine aminotransferase in QD800-GRD two injection group were decreased;but the differences were not statistically significant between four groups (P>0.05). Compared with PBS injection group,the activities of SOD,GSH and the MDA contents in liver,spleen,kidney and lung tissues in QD800-RGD one injection group and QD800-RGD two injection group were slightly increased;but the differences were not statistically significant between four groups (P>0.05). Compared with PBS injection group,the morphology of liver,spleen,kidney and lung tissues in QD800-RGD one injection group and QD800-RGD two injection group were normal,the cells agganged orderly, and no necrosis was seen. Conclusion At the dose needed to produce in vivo tumor imaging,there is no significant toxicity of NIR QD800-RGD in the mice after repeatedly intravenous injection.

Objective To explore the effects of Juglone on the proliferation and cell cycle of human cervical cancer SiHa cells, and to clarify the therapeutic effect of Juglone in cervical cancer. Methods The SiHa cells at logarithmic phase were divided into control group and different doses (10,20,40,60,80,100 μmol·L^-1) of Juglone groups.The morphological changes of the SiHa cells were observed under inverted phase contrast microscope.The proliferation activities and the inhibitory rats of growth of SiHa cells were measured by MTT assay.The cell cycle of SiHa cells was detected by flow cytometry (FMC). Results Compared with control group,the volume of SiHa cells reduced,the connection with the surrounding cells disappeared,and they detached from the surrounding cells in Juglone groups.Compared with control group,the proliferation activities of the SiHa cells in Juglone groups were decreased (P<0.05) and the inhibitory rates of growth of SiHa cells were increased in a dose-dependent manner in Juglne groups; the IC₅₀ value of Juglone was 20.7 μmol·L^-1.The results of FMC showed that the ratios of SbuG₁ state in Juglone groups were increased,and the ratios of SiHa cells at G₀/G₁ and S phases were decreased,and the ratios at G₂/M phase were increased previously then were decreased in 10 and 100 μmol·L^-1 Juglone groups compared with control group. Conclusion Juglone could inhibit the proliferation of SiHa cells and cause the increase of the SubG₁ ratio of cell cycle.

Objective To investigate the effect of FZD4 on the self-renewal ability of glioma stem cells,and to clarify its mechanism. Methods Two shRNA sequences were designed according to FZD4 mRNA sequence,named FZD4-shRNA-1# and FZD4-shRNA-2#.Then the lentiviral vector particles were transfected into U251cells(FZD4-shRNA-1# and FZD4-shRNA-2#),The U251 cells infected with the empty vector were used as control group.The expression of green fluorescence protein (GFP) in the transfected cells were observed under inverted fluorescence microscope.The expression levels of FZD4 mRNA and protein in the U251 cells in various gorups were detected by reverse transcription (RT)-PCR and Western blotting method.The U251 cells were cultured in SFM and the glioma stem cells were selected;the proliferation and self-renewal of the tumore stem cells were observed with limiting dilution assay. Results Compared with control group,the expression levels of FZD4 mRNA and protein in FZD4 shRNA groups were significantly reduced(P<0.01).The diameters of neurospheres in FZD4 shRNA groups were significantly smaller than that in control group(P<0.01).The number of cells required to generate at least one tumor sphere/well was significantly increased in FZD4 shRNA groups than that in control group(P<0.05 or P<0.01) in limiting dilution assay. Conclusion Down-regulation of FZD4 expression that in inhibit the self-renewal ability U251 glioma stem cells.

Objective To discuss the influence of the changes of estrogen receptor-alpha (ERα) promoter methylation in the expression levels of MTA1 mRNA and protein in breast cancer cell lines,and to illustrate the effect of ERα promoter on the expression of MTA1 gene after demethylation. Methods The breast cancer cell lines MDA-MB-435s in ERα promoter in hypermethylation status and MDA-MB-231 in ERα promoter in hypomethylation status were treated with 5-Aza-2'-deoxycytidine (5-Aza-dC) (5-Aza-dc treated groups),while the two kinds of cell lines were without 5-Aza-dC treatment were used as untreated groups.The methylation status of ERα promoter of the cells in various groups was detected by methylation-specific PCR (MSP).The expression levels of MTA1 mRNA and protein in MDA-MB-435s and MDA-MB-231 cells in various groups were detected with RT-PCR and Western blotting method. Results Both in the MDA-MB-435s and the MDA-MB-231 cells,the methylation status of ERα was modified from methylation to unmethylation by being treated with 5-Aza-dC.Furthermore,both in the MDA-MB-435s and the MDA-MB-231,compared with untreated groups,the expression levels of MTA1 mRNA and protein in 5-Aza-dC treated groups were down-regulated (P<0.05). Conclusion After the methylation status of ERα promoter is modified from methylation to unmethylation in breast cancer cell lines MDA-MB-435s and MDA-MB-231,the expression levels the MTA1 mRNA and protein are down-regulated.It's illustrated the expression of MTA1 can be lowered after ERα promoter demethylation,and the results further verify the regulation of the relationship between ERα methylation and MTA1 gene.

Objective To investigate the influence of erythropoietin(EPO) in the bone defect after localized on polylactic-co-glycolic acid(PLGA) surface. Methods The bone marrow stromal cells (BMSCs) were isolated and cultured,and the adenovirus vector AdCMV-EGFP was used for gene transfection. The lyophilization method was used to compound AdCMV-EGFP and PLGA nanofiber scaffold as experiment group(PLGA/Ad-EGFP group),and the equivalent Ad-EGFP as control group;the adenovirus viability was detected using the percentage of fluorescence cell area of BMSCs infected with EGFP.Quantitative Picogreen dsDNA kit was used to detect the release kinetics of adenovirus.The Ad-EPO and PLGA nanofiber scaffold was compounded as experiment group(PLGA/Ad-EPO group),and single bone defect as control group;the PLGA/Ad-EPO were implanted into the critical skull bone defect of the rats,and the percentages of new bone areas at the 4th week and 8th week were detected by muro-CT and HE staining. Results Compared with control group,the percentage of fluorescentce cell area of the rats in PLGA/Ad-EGFP group was increased(P<0.05).The release kinetics of adenovirus on PLGA was a burst release curve,the retention on PLGA of adenovirus in the first hour was 53.0%±5.6%,and in the 16th hour was 20.0%±3.3%. Compared with control group, the erythrocytopoiesis was promoted at the 4th week, and the percentages of new bone areas at the 4th and 8th week were increased to 25.0%±5.7% and 35.0%±6.3% in PLGA/Ad-EPO group(P<0.05). Conclusion The lyophilizaion method could reserve the adenovirus activity,and PLGA/Ad-EPO nanofiber scaffold could promote the bone defect repair in a certain extent.

Objective To study the role of mTORC2/SGK1 signaling pathway in the up-regulation of alveolar epithelial sodium channel α-subunit (α-ENaC) by insulin,and to clarify the mechanism of insulin in promoting the lung edema clearance in the acute lung injury(ALI) mice. Methods The C57BL/6J mice were randomly divided into control group,LPS group,insulin group (LPS+insulin),PP242 group (PP242+LPS+insulin) and rapamycin group (rapamycin+LPS+insulin),with 10 mice in each group. The lung wet/dry weight(W/D) ratios and alveolar fluid clearance(AFC) of the mice were detected. HE staining was used to observe the pathological changes of lung tissue. The protein expression levels of α-ENaC and phosphorylated serum-and the levels of glucocorticoid-inducible kinase 1 (SGK1) at Ser422 in lung tissue were determined by Western blotting method. Results Compared with LPS group, the lung injury score and W/D ratio in insulin group were decreased significantly (P<0.05) and the AFC was increased significantly (P<0.05).The expression level of α-ENaC protein in LPS group was decreased significantly compared with control group (P<0.05). The expression levels of both α-ENaC protein and pSGK1(Ser422) in insulin group were significantly increased compared with LPS group (P<0.05).Compared with insulin group,the α-ENaC protein expression level and phosphorylated SGK1(Ser422) levelin PP242 group were decreased(P<0.05). Conclusion Through mTORC2 pathway,insulin activates the SGK1 and up-regulates the expression of α-ENaC protein to accelerate the AFC,which is beneficial to the prognosis of ALI/acute respiratory distress syndrome(ARDS).

Objective To observe the regulation effect of spinal cord stimulation (SCS) on the cardiac nociception of the rats induced by bradykinin (BK), and to explore its mechanism. Methods 42 male healthy SD rats were randomly divided into electrophysiological experiment part (N=17) and immunohistochemistry part (N=25).All the rats were implemented cardiac catheterization to evoke heart pain.In electrophysiological experiment part,the rats were divided into BK reproducibility group (n=5) and BK+SCS group (n=12),and the electromyogram (EMG) percentage changes of dorsal spinotrapezius muscle evoked by BK were used as study indexes to examine the descending modulation of SCS on the cardiac nociception.In immunohistochemistry part,the rats were divided into control group (n=3),saline group (n=4),SCS group (n=6),BK group (n=6) and BK+SCS group (n=6).The expression level of c-fos in T3-T5 spinal dorsal horn was used as nociceptive evaluation to assess the protective effect of SCS on the cardiac nociception. Results Compared with BK reproducibility group,the EMG percentage in BK+SCS group was decreased (P<0.05),and the EMG declined to (54.02±4.95)% of control at 5 min after SCS (P<0.05);at 55 min after SCS,the EMG recovered to (74.82±4.74)% of control (P<0.05) and 105 min later,the EMG recovered to control level. Moreover,compared with control group (15.00±2.87),the expression levels of c-fos in saline control group (22.50±1.85) and SCS group (35.00±3.77) had no significant differences (P>0.05). The expression levels of c-fos in BK group (115.67±10.05) in T3-T5 spinal cord dorsal horn was higher than that in control group (P<0.05), and the expression level of c-fos (59.83±5.76) in BK+SCS group was significantly lower than that in BK group (P<0.05). Conclusion SCS can significantly inhibit the EMG and c-fos expression level induced by BK,which may be attributed to that SCS activates the spinal inhibitory interneurons and these inhibitory interneurons suppress the activities of cardiac pain sensitive neurons.

Objective To detect the expression of DNA dioxygenase Ten-Eleven Translocation 2 (TET2) in the brain tissue of Alzheimer's disease(AD) animal models,and to explore the protective effect of TET2 protein on the neurons under oxidative stress. Methods The C57BL/6 mice were divided into adult group,old age group and AD group,which were 8 weeks,8 months normal mice and 8 months Alzheimer's transgenic mice, respectively.The TET2 mRNA levels in the cerebral cortex,hippocampus and cerebellum tissue of the mice in three groups were examined using Real-time quantitative PCR.The hippocampal neuron cell lines HT22 of mice were randomly divided into normal group,control group,lentivirus control group (N.C.shRNA) and lentivirus-mediated TET2 interference groups (TET2 shRNA-1 and TET2 shRNA-2).After 72 h of lentivirus infection,the HT22 cells in control group,N.C.shRNA group and two TET2 shRNA groups were given 50 μmol·L^-1 hydrogen peroxide (H₂O₂) and 2.5 mmol·L^-1 L-homocysteic acid (L-HCA) for 4 h to establish the extracellular and intracellular oxidative stress models,then the viabilities of the cells in various groups were determined with MTT method. Results With the increase of age,the TET2 mRNA in the cortex,hippocampus and cerebellum were increased markedly. Compared with old age group,the TET2 mRNA expression levels in the cortex,hippocampus and cerebellum tissue of the mice in AD group were significantly decreased (P<0.05 or P<0.01). The survival rate of the neurons in TET2 shRNA-2 group was significantly lower than that in N.C shRNA group in H₂O₂ and L-HCA mediated oxidative stress models (P<0.01). Conclusion The expression level of TET2 in brain tissue of the AD mice is notably reduced,and the down-regulation of TET2 causes the neurons to be more susceptible to oxidative damage,which implicating that TET2 maybe protect the neurons against oxidative stress.

Objective To study the effects of SiO₂ nanoparticles (SiO₂ NPs) on the cell adhesion and migration of HL-7702 cells, and to provide experimental basis for safety evaluation of SiO₂ NPs. Methods The in vitro cultured human normal liver cell line HL-7702 were randomly divided into control and SiO₂ NPs exposure groups at the concentrations of 12.5,25.0,50.0,and 100.0 mg·L^-1,respectively.Transmission electron microscope (TEM) and dynamic light scattering (DLS) method were used to observe the characteristics of SiO₂ NPs.After 24 h exposure,cell adhesion assay was applied to examine the changes of cell adherence;wound healing assay was used to discover the changes of cell migration;the cellular uptake and biodistribution of SiO₂ NPs were observed using TEM. Results Based on the result of TEM,SiO₂ NPs were spherically shaped,uniformly sized and sporadically dispersed with the diameter of (67.42±5.69) nm. The results of DLS method showed that the average hydrated particle size of SiO₂ NPs dispersing in RPMI-1640 serum-free medium was (134.13±2.78) nm,while the hydrated particle sizes of SiO₂ NPs dispersing in RPMI-1640 with 1%,5%,or 10% serum were apparently enlarged.The results of cell adhesion and wound healing assays showed that the adhesion rates and the healing rates of cells could be down-regulated by SiO₂ NPs even under a dose that did not affect the cell viability and membrane integrity(P<0.05),furthermore,the down regulation effect was significantiy increased along with the enlargement of the SiO₂ NPs' concentration. The TEM results showed that after exposed to nontoxic dose of SiO₂ NPs,the cells accumulated a large number of nanoparticles,the particles mostly dispersed in endocytic recycling compartment,and some were in the cytoplasm;some parts of the cell membrans were caved,and the procedure of endocytose could be captured. Conclusion SiO₂ NPs could inhibit the cell adhesion and migration in a dose-dependent manner.

Objective To explore the effect of Vitacamphorae Injection on the myocardial fibrosis induced by isoproterenol(Iso) in the rats,and to clarify its mechanism of anti-myocardial fibrosis,and to provide the basis for its clinical application. Methods 60 SD rats were randomly divided intocontrol group,Iso model group, vitamin C group and different doses of Vitacamphorae Injection groups. The models of myocardial fibrosis in the rats were set up by using subcutaneous injection of 2 mg·kg^-1·d^-1 Iso,for 14 consecutive days,while the rats in different doses of Vitacamphorae Injection groups and vitamin C group were subcutaneouly injected with 0.8,1.6,3.2 mg·kg^-1·d^-1 vitacamphorae and 80 mg·kg^-1·d^-1 vitamin C,for 28 consecutive days. BL-420 E⁺ biological function experimental system was used to detect the rat ECG and heart function after experiment. Thenthe heart mass index (HMI) and left ventricular mass index (LVMI) were measured. The contents of malondialdehyde (MDA),the activities of superoxide dismutase (SOD) and inducible nitric oxide synthase enzyme (iNOS)in the myocardium tissue were measured by UV detection.The activities of the lactate dehydrogenase (LDH) and the contents of nitric oxide (NO) were examined by enzyme labelling instrument.The contents of type Ⅰ (ColⅠ) collagen and type Ⅲ collagen (Col Ⅲ) in the myocardium tissue of the rats were detected by ELISA.The pathological changes of myocardium tissue were observed by HE staining. Results Compared with control group,the heart rate of the rats in Iso model group was increased,the left ventricular systolic pressure (LVSP) was decreased,and the maximum rising rate of left ventricular pressure (LVdp/dt_max) was decreased (P<0.05 or P<0.01); the HMI and LVMI were increased,but there were no significant differences(P>0.05).In left ventricular myocardium tissue,the contents of MDA,Col Ⅰ and Col Ⅲ were increased,while the NO content was decreased(P<0.05 or P<0.01),and the activities of SOD and iNOS were decreased,but the LDH activity was increased(P<0.05 or P<0.01).Compared with Iso model group,the HMI and LVMI of the rats in different doses of Vitacamphorae Injection groups were decreased in a dose-dependent manner;and the contents of MDA,the LDH activities,the Col Ⅰ and Col Ⅲ contents were decreased(P<0.05 or P< 0.01),while the NO contents and the activities of iNOS,SOD were increased(P<0.05 or P<0.01). Conclusion Vitacamphorae Injection could inhibit the myocardial fibrosis induced by Iso in the rats,and its mechanism may be related to antioxidant and increasing the level of NO to inhibit the synthesis of collegen.

Objective To investigate the synergistic effect of sunitinib combined with NK cells on the renal clear cell carcinoma 786-0 cells,and to clarify the mechanism. Methods The 786-0 cells were cultivated by routine method in vitro and divided into untreated group and sunitinib group,meanwhile positive control group (K562 cells) was set up.The human NK cells were isolated by magnetic activated cell sorting (MACS).The sensitivity of 786-0 cells to sunitinib was analyzed by MTT assay.Flow cytometry was used to evaluate the purity of isolated cells and the expression rates of NKG2D-ligands (NKG2DLs) on the target cells before and after treatment of sunitinib.Subsequently,the cytotoxic sensitivities of 786-0 cells to NK cells in untreated group,positive control group and sunitinib group were measured by LDH releasing assay. Results The IC₅₀ values of sunitinib for 786-0 cells was (4.45±0.65) μmol·L^-1.More than 72% of isolated NK cells showed to be CD3^-CD16⁺CD56⁺ cells which would definitely meet the needs of experiments. The expressions rates of MICA,MICB,ULBP2 on the target cells incubated with sunitinib were respectively increased from (3.52±0.29)%,(3.10±0.73)%,(5.12±4.77)% to (15.45±2.14)%,(21.95±3.08)%,(51.71±5.33)%.Compared with untreated group,the expression rates of MICA,MICB and ULBP2 in treatment group were significantly increased(P<0.05).At the E:T ratio of 10:1 and 20:1,the cytotoxic sensitivities of 786-0 cells to NK cells were increased from (9.71±0.88)% and (21.28±0.32)% in untreated group to (20.83±1.28)% and (35.11±0.70)% in sunitinib group(F＝166.18,P＝0.000;F=52.87,P=0.000);the cytotoxic sensitivities of NK cells to the 786-0 cells in sunitinib group were higher than that in untreated group (P<0.05). Conclusion Sunitinib can selectively regulate the expressions of NKG2DLs (MICA/B and ULBP2) in the 786-0 cells,which can result in higher cytotoxic sensitivity to NK cells.Sunitinib combined with NK cells has synergistic effect on the renal clear cell carcinoma cells.

Objective To investigate the changes of the expression of apelin-APJ system in the injury of pulmonary vascular endothelial cells (PVECs) under hypoxia conditions, and to clarify the role of apelin in the cell proliferation. Methods The in vitro cultured rat PVECs were divided into control group, hypoxia 6 h group, hypoxia 12 h group,hypoxia 24 h group,hypoxia 48 h group,hypoxia 24 h + apelin group and apelin group. The proliferation rates of PVECs in every group were detected by MTT assay. The in vitro cultured rat PVECs were divided into normal control group,hypoxia 6 h group,hypaxia 12 h group and hypoxia 24 h group.The expression of apelin in the cells were observed by immunofluorescence staining. The expression levels of apelin-APJ protein were measured by Western blotting method. Results Compared with normal control group, the proliferation rates of the PVECs in hypoxia 6 h,12h groups and apelin group had no change(P>0.05), but the proliferation rates of the PVECs in hypoxia 24 h and 48 h groups were decreased significantly(P<0.05 or P<0.01). Compared with normal control group, the immunofluorescence expression of apelin in the PVECs in hypoxia 6 h group had no obvious change(P>0.05), but the expression of apelin in the PVECs in hypoxia 12 h and 24 h groups were decreased (P<0.01). Compared with normal control group, the expression levels of apelin protein in the PVECs in hypoxia 6 h, 24 h and 48 h groups were decreased(P<0.01), but the protein expression of APJ in the PVECs had no significant changes(P>0.05). Conclusion Hypoxia may injury the PVECs through inhibiting the apelin expression, and exogenous apelin can protect the injure of PVECs induced by hypoxia.

Objective To determine the levels of lipopolysaccharide (LPS) in portal vein blood from the chronic intake rats,and to explore the mechanism and significance of LPS-Toll-like receptor 4 (TLR4) pathway in hepatic fibrogenesis in the rat models of chronic alcohol intake. Methods Twenty-four male Sprague-Dawley rats were randomly divided into alcohol group and control group,and the rats were fed with isocaloric Lieber-Decarli alcohol liquid diets and control diets for 10 weeks.The histological characteristics of liver tissue of the rats were evaluated by HE staining and Masson Trichrome staining.The plasma LPS levels in portal veins of the rats were determined by spectrophotometric method. The fibroblast specific protein 1(FSP-1) and vimentin were determined for identification of hepatic stellate cells (HSCs) by immunohistochemistry.The TLR4 mRNA expression levels in rat liver tissue were examined by in situ hybridization method.Computer image analysis was performed to measure the integrated optimal density (IA) of FSP-1-positive HSCs or TLR4 mRNA-positive cells,respectively.The expression levels of interleukin 1(IL-1),interleukin 6(IL-6),tumor necrosis factor α(TNF-α) and transforming growth factor beta 1(TGF-β1) mRNA in liver tissue were detected by RT-PCR. Results Compared with control group, the liver tissue of the rats in alcohol group showed a mild perisinusoidal and periportal fibrosis;the expression level of TLR4 mRNA and the number of FSP-1-positive HSCs were increased at the end of 10 weeks (P<0.05).At the end of 10 weeks,the plasma LPS level in portal veins of the rats in alcohol group was significantly higher than that in control group (P<0.01).Compared with control group,the expression levels of IL-1,IL-6,TNF-α and TGF-β1 mRNA in liver tissue of the rats in alcohol group were increased significantly(P<0.01).Bivariate correlation analysis showed that there was a positive correlation between the expression intensity of TLR4 mRNA in liver tissue and the levels of portal vein plasma LPS of the rats in alcohol group (r=0.856,P<0.05). Conclusion The activation of TLR4 pathway triggered by chronic intake plays an important role in the rat hepatic fibrogensis through up-regulation of profibrogenic and proinflammatory cytokine release.

Objective To study the role of liver total histone epigenetic modification changes in the development of type 2 diabetes mellitus in the mice,and to explore its clinical significance. Methods Thirty C57BL/6J mice were divided into normal group,high-fat high-sucrose (HFS) group and type 2 diabetes mellitus (T2DM) group.The mice in normal and HFS groups were given regular diet and HFS diet,respectively,while type 2 diabetic mice were induced by HFS diet plus streptozotocin (STZ).HE staining,Western blotting,Quantitative PCR and ChIP assay were used to analyze the pathological changes of liver tissue of the mice,total histone H3 modification,the expression levels of glucose metabolism-related genes and histone H3 modification in gene promoter. Results The liver pathology after HE staining and modification patterns of histone of the mice in T2DM group were significantly different from other groups.Compared with normal group,the H3K23 acetylation of the mice in HFS group was decreased (P<0.01),the modification levels of H3K9Ac and H3K9Me2 were increased (P<0.01),the level of H3K4Me had no change (P>0.05),and the expression levels of glucose metabolism-related genes Pklr,Glut2 and Gck were increased (P<0.01). Compared with normal group,the modification levels of H3K23Ac (P<0.01) and H3K9Ac (P<0.05) in T2DM group were decreased, the modification levels of H3K9Me2 and H3K4Me were increased (P<0.01),and the expression levels of glucose metabolism-related genes Pklr,Glut2,Gck,Ppargc1a and Insr were decreased (P<0.05 or P<0.01).Moreover,the changes of H3K23Ac,H3K9Ac,H3K9Me2 and H3K4Me modification in gene promoter of Glut2 and Pklr showed consistent with its expression levels. Conclusion The epigenetic modification changes of liver histone might participate in the occurrence and development of type 2 diabetes mellitus.

Objective To observe the influence of different doses of 17β-estradiol (17β-E2) on the osteogenesis differention of bone marrow mesenchymal stem cells (MSCs) and the expression of bone gamma-carboxyglutamic-acid-containing proteins (BGP) gene, and to clarify the mechanism of estrogen replacement treatment for postmenopausal osteoporosis. Methods The MSCs were isolated by density gradient centrifugation and adherence screening method for continuous three passages,and the osteogenic induction and differention were performed. The MSCs were divided into control group(OBM) and different doses of 17β-E2 groups(anding 0.1,1.0,10.0 and 100.0 pmol·L^-1 17β-E2 in osteogenic induction liquid).On the 7th day,radioimmunoassay was used to detect to alkaline phosphatase (ALP) activity,Von Kossa staining for calcium deposition of the cells in various groups.On the 14th day,the collagenⅠ (ColⅠ) level was determined by enzyme linked immunosorbent assay.On the 21th day,the BGP mRNA expression level was detected by real-time fluorescence quantitative PCR,and the cells'optical density (A) at 560 nm was determined. Results The MSCs were isolated successfully by density gradient centrifugation and adherence screening method.The MSCs appeared fibroblasts-like with ellipse nucleus with one to two nucleolus.The subculturing MSCs grew well and maintained the morphological characteristics of primary cells.Compared with control group,the ALP activities and Col Ⅰ levels in different doses of 17β-E2 groups were increased(P<0.05 or P<0.01),and the bone matrix calcification was enhanced in a dose-dependent manner (P<0.05 or P<0.01);the BGP mRNA expression level in 100.0 pmol·L^-1 17β-E2 group was increased(P<0.01). There were significant differences in the BGPmRNA expression levels difference between 10.0 and 100.0pmol·L^-1 groups (P<0.05) ALP. The col-I levels and bone matrix calcification had significant differences between various groups (P<0.05). Conclusion The separated cells in this study are the rat MSCs. 17β-E2 can enhance the osteogenesis differention of MSCs and up-regulate the BGP mRNA expression in a dose-dependent manner.

Objective To observe the improvement effect of Astragalus(AS) Injection on the ventricular remodeling in the rats after myocardial infarction and its regulation effect on the protein kinase C (PKC), and to clarify the mechanisms. Methods The left anterior descending(LAD) branch of coronary artery of 42 healthy Wistar rats were ligated in order to establish ventricular remodeling model after myocardial infarction.36 rats survived after 24 h were divided into three groups and 12 in each group. The rats in sham operation group were given physiological saline 2 mL·d^-1 by intraperitoneal injection; the rats in myocardial infarction group (model group) were given physiological saline 2 mL·d^-1 by intraperitoneal injection;the rats in AS group were treated with AS Injection 6 g·d^-1 (2 mL·d^-1) by intraperitoneal injection.After 6 weeks the survival situation of the rats in various groups was observed.The echocardiographic examination was made to inspect the left ventricular end diastolic diameter (LVEDD),left ventricular end systolic diameter (LVESD),left ventricular ejection fraction (LVEF) and left ventricular fraction shortening (LVFS).The levels of plasma brain natriuretic peptide (BNP) of the rats in various groups were detected.Masson staining was used to determine the collagen volume fraction (CVF) of myocardium and the PKCα and PKCε expression levels in myocardial cells were detected by immunohistochemical staining and Western blotting method. Results The number of dad rats in AS group was less than that in model group.Compared with sham operation group,the LVESD and LVEDD of the rats in model group were increased (P<0.05),the LVEF and LVFS were decreased (P<0.05),and the BNP lelvel was increased (P<0.05). Compared with model group,the LVESD and LVEDD of the rats in AS group were decreased (P<0.05),the LVEF and LVFS were increased (P<0.05),and the BNP level was decreased (P<0.05).Compared with sham operation group,the CVF in the myocardial cells of the rats in model group was increased(P<0.05). Compared with model group,the CVF in the myocardial cells of the rats in AS group was decreased (P<0.05). Compared with sham operation group,the PKC expression level in the myocardial cells of the rats in model group was increased significantly (P<0.05). Compared with model group,the PKCα expression level in the myocardial cells of the rats in AS group was decreased significantly (P<0.05),and the PKCε expression level was increased significantly (P<0.05). Conclusion AS Injection can improve the ventricular remodeling in the rats after myocardial infarction,and its mechanism may be related to up-regulating the expression of PKCε and inhibiting the expression of PKCα.

Objective To observe the influence of SF/PLLA composite fibrous scaffolds with different quality ratios of SF and PLLA in the proliferation and phenotypes of epithelial cells and fibroblasts, and to clarify the cytocompatibility of SF/PLLA bioengineering membrane. Methods The SF and PLLA were served as materials to construct composite scaffolds.The different proportions of SF/PLLA nanofibers membrane were compounded with different ratios of SF and PLLA(S50/P50,S40/P60 and S30/P70) and mixed spinning.The primary epithelial cells and fibroblasts from rat skin tissue were cultured on the SF/PLLA composite scaffolds.CCK8 test was used to evaluate the cell proliferation,and immunofluorescence staining was used to determine the expressions of the specific marker proteins of cultured cells. Results The proliferation activities of fibroblasts in S50/P50 group were significantly higher than those in S40/P60 and S30/P70 groups,and the proliferation activities of epithelial cells on the composite scaffolds in S40/P60 group were higher than those in S50/P50 and S30/P70 groups (P<0.01) on the 5th and 6th days.The epithelial cells cultured on the SF/PLLA composite scaffolds expressed CK 19 protein and the fibroblasts expressed Vimentin protein.When two kinds of cells were co-cultured on SF/PLLA composite fibrous scaffolds,the cell growth status in S40/P60 and S50/P50 groups were superior to that in S30/P70 group. Conclusion The increasing of SF concentration in composite scaffolds can obviously increase the cell proliferation activity and promote the cell survival;SF/PLLA scaffolds has no significant influence in the cell phenotype.

Objective To investigate the inhibitory effects of different Ginkgo biloba leaf extract (GBE) ingredients on the proliferation and extracellular matrix (ECM) accumulation of high glucose-treated mesangial cells,and to clarify their mechanisms. Methods The high glucose-treated mesangial cells (HBZY-1) were given GBE,total flavonoids,total ginkgolide,total flavonoids hydrolyzate,quercetin,kaempferol,isorhamnetin,ginkgolide A,B,and ginkgolide C; the concentrations were 0(high glucose control group),3.125,6.250,12.500,25.000 and 50.000 mg·L^-1,meanwhile low glucose control group was set up. The cell proliferation was detected by MTT assay. The high glucose-treated mesangial cells were given GBE,total flavonoids,total ginkgolide,ginkgolide A,ginkgolide B,and ginkgolide C;the concentrations were 0(high glucose control group),0.05,0.50,5.00,50.00 mg·L^-1,meanwhile low glucose control group was set up. The levels of fibronectin(FN) and type Ⅳ collagen (Col Ⅳ) were detected by ELISA method. Results The MTT results showed that compared with high glucose control group,the proliferation activity of mesangial cells in 50.00 mg·L^-1 GBE group was decreased significantly (P<0.01);the proliferation activities of mesangial cells in 25.00 and 50.00 mg·L^-1 total flavonoids group were decreased significantly(P<0.05 or P<0.01);the proliferation activities of mesangial cells in all quercetin,kaempferol,isorhamnetin and total flavonoids hydrolyzate groups were decreased significantly (P<0.01);the proliferation activity of mesganial cells in 50.00 mg·L^-1 ginkgolide C group was decreased significantly (P<0.01). The ELISA results showed that compared with high glucose control group,the levels of Col Ⅳand FN in 5.00 and 50.00 mg·L^-1 GBE groups were decreased significantly (P<0.01),and the FN level in 0.50 mg·L^-1 GBE group was decreased significantly (P<0.05);the Col Ⅳ levels in 0.50,5.00,and 50.00 mg·L^-1 total flavonoids groups were decreased significantly(P<0.05 or P<0.01);the Col Ⅳand FN levels in all total ginkgolide groups had no obviously differences except 50.00 mg·L^-1ginkgolide C group (P<0.05 or P<0.01);the FN level in 50 mg·L^-1 ginkgolide C group was decreased significantly(P<0.05). Conclusion GBE can inhibit the high glucose-induced proliferation only in the high concentration (50 mg·L^-1) and inhibit the high glucose-induced mesangial cell ECM accumulation in the low concentration (0.50-25.00 mg·L-1);the total flavonoids plays a major role in the inhibition of cell proliferation and ECM accumulation,and the total ginkgolide has no significant effect;the inhibitory effect of aglycones on mesangial cell proliferation is much stronger than that of glucoside.

Objective To explore the cytotoxicity of anthocyanins from purple sweet potato on the human colorectal carcinoma cells (HCT-116),and to illuminate the mechanism of inducing apoptosis. Methods The HCT-116 cells and fibroblasts HF-91 at the logarithmic phase were divided intocontrol group(PBS),positive drug group (25 mg·L^-1 DDP),PCI groups (final concentrations:25,50,and 100 mg·L^-1). The inhibitroy rates of cell proliferation were detected with CCK8 assay;the apoptosis was observed using AO/EB method;the JC-1 mitochondrial membrane potential was detected by flow cytometry; the expression levels of p53 and caspase-3 mRNA were measured by qPCR method. Results Compared with DDP group,the inhibitory rate of proliferation of HCT-116 cells in 100 mg·L^-1 PCI group was increased(P<0.05),and the inhibitory rates of proliferation of HF-91 cells in different concentraitons of PCI groups were decreased(P<0.05) in a concentration-dependent manner.Compared with DDP group,the apoptotic rates of HCT-116 cells in 50 and 100 mg·L^-1 PCI groups were increased(P<0.05).Compared with control group,the expression levels of p53 and caspase-3 mRNA in HCT-116 cells in DDP group and different concentrations of PCI groups were increased(P<0.05).Compared with DDP group,the expression levels of caspase-3 mRNA in different concentrations of PCI groups were significantly increased(P<0.05),and the expression level of p53 mRNA in 100 mg·L^-1 PCI group was obviously increased(P<0.05). Conclusion Proanthocyanidin has strong cytotoxicity on the digestive system tumor cells,and has little cytotoxicity on the normal cells.

Objective To investigate the effects of transforming growth factor β1 (TGF-β1) on the epithelial-to-mesenchymal transition(EMT)in human tongue squamous cell carcinoma, and to explore its influence in PI3K/AKT signaling pathway. Methods The in vitro cultured tongue squamous cell carcinoma SCC9 cells and CAL27 cells were divided into control group and different concentrations (5 and 10 μg·L^-1)of TGF-β1 groups;24 h after TGF-β1 treatment,the morphology of cells in various groups were observed under light microscope. Immunoflurescence staining was used to detect the expressions of Vimentin and E-cadherin in the cells in various groups. The expressions of AKT and p-AKT in the SCC9 and CAL27 cells were measured by Western blotting method. Results Compared with control group,the SCC9 and CAL27 cells with polygonal morphology trans-differentiated into mesenchymal phenotype with an elongated morphology in TGF-β1 groups.Compared with control group,the expressions of Vimentin protein in the cells in TGF-β1 groups were increased and the expressions of E-cadherin were decreased. Compared with control group,the expression levels of p-AKT were decreased in SCC9 and CAL27 cells in TGF-β1 groups. Conclusion TGF-β1 can induce the EMT in human tongue squamous cell carcinoma and affects the PI3K/AKT signaling pathway.

Objective To study the caveolin-3(CAV3) protein expression in human colorectal cancer (CRC) tissue,and the effect of cantharidin on CAV3 expression,and to clarify the molecular mechanism of cantharidin in the treatment of CRC. Methods A total of 30 surgically removed CRC tissue were selected, meanwhile,the adjacent mormal tissue was collected and used as normal control group.HE staining and immunohistochemical method were used to detect the positive expression rate of CAV3 in the human CRC tissue.The human CRC HCT-116 cells were divided into control group,fluorouracil treatment group and cantharidin treatment group.The expression levels of CAV3 mRNA were detected by Real-time PCR and immunohistochemistry staining. Results The results of HE showed that the nuclei of tumor cells were stained deeply and arranged tightly.There were 22 cases among 30 cases with positive CAV3 expression,and its positive expression rate was 70.3%.The immunohistochemistry staining results showed that the number of HCT-116 cells in cantharidin group was decreased compared with control group(P<0.01);the expression levels of CAV3 in the HCT-116 cells in fluorouracil and cantharidin groups were decreased compared with control group (P<0.05).The Real-time PCR results showed that the CAV3 mRNA levels in the HCT-116 cells in fluorouracil and cantharidin groups were significantly decreased compared with control group(P<0.05). Conclusion The CAV3 protein highly expresses in the majority of human CRC tissue.Cantharidin might inhibit the growth of CRC cells through down-regulatiing the CAV3 expression.

Objective To explore the effects of hepatocyte growth factor (HGF) and angiotensin Ⅱ (Ang Ⅱ) on the tubular epithelial myofibroblasts (TEMT) transdifferentiation,and to clarify their mechanisms. Methods The cultured human proximal convoluted tubule HK-2 cells in vitro at stage Ⅳ were divided into control group,AngⅡ(1×10^-6mol·L^-1) group,HGF(8 μg·L^-1)group,AngⅡ(1×10^-6mol·L^-1)+HGF(8 μg·L^-1)group.The proliferation of HK-2 cells was detected by CCK8 method;the expressions of α-smooth muscle actin (α-SMA) mRNA were detected by RT-PCR and the α-SMA protein expression levels were detected by Western blotting method. Results Compared with control group,the proliferation activity of cells in AngⅡ group was decreased (P<0.01),the proliferation activity of the HK-2 cells in HGF group was increased(P<0.05),and it was also decreased in AngⅡ+HGF group (P<0.05).Compared with control group,the α-SMA mRNA and protein expression levels in the HK-2 cells in AngⅡ group were increased (P<0.05 or P<0.01), and the expression levels of α-SMA mRNA and protein in HGF group were decreased (P<0.05 or P<0.01).Compared with AngⅡ group,the proliferation activity of HK-2 cells were increased (P<0.05),and the α-SMA mRNA and protein expression levels in AngⅡ+HGF group were decreased (P<0.05). Conclusion AngⅡ can inhabit the proliferation of HK-2 cells and promote TEMT.HGF can promote the proliferation of HK-2 cells and inhabit TEMT.HGF may interfere the effects of AngⅡ on inhibiting the proliferation of HK-2 cells and promoting TEMT.

Objective To investigate the association between the single nucleotide polymorphisms(SNPs)of DNA methyltransferase 1(DNMT1) gene and the survival of the patients with gastric cancer after operation in the Han population from North China,and to clarify the association between the polymorphisms of DNMT1 gene and prognosis of gastric cancer. Methods A total of 447 gastric cancer patients treated with total gastrectomy were selected into the cohort study.The distribution of genotypic frequencies of five SNPs sites(rs16999593,rs10420321,rs2288349,rs2228611 and rs2228612)of DNMT1 were detected by TaqMan assay.447 cases of gastric cancer patients were followed up.Kaplan-Meier method was used to calculate the survival and draw the survival curves,and Log-rank test was used to compare the differences of overall survival(OS)between the patients with different genotypes.The hazardratio (HR)and 95% confidence interval(CI)were estimated by univariate and multivariate Cox regression analysis. Results Compared with the patients carried GG genotype at the rs2228611 site,the patients with GA/AA genotypes had better prognosis and Longer survival time(52.5 months vs 42.9 months,Log-rank P=0.006).After adjusting the confounding factors,the patients carried rs2228611 GA/AA genotypes had a decreased death risk than the patients with GG genotype(HR 0.68,95% CI:0.50-0.92,P=0.013),suggesting that the polymorphisms of rs2228611 site was an independent influencing factor of prognosis of the patients with gastric cancer after operation. There was no association between the polymorphisms of other sites and the prognosis of gastric cancer. Conclusion The polymorphisms of DNMT1 rs2228611 GA/AA genotypes can prolong the survival time of the patients with gastric cancer.The polymorphism of DNMT1 could be used as a biomarker to predict the prognosis of gastric cancer.

Objective To investigate the relationships between apolipoprotein E(APOE) genotypes and blood lipid levels in the male population,and to provide theoretical basis for the diagnosis of dyslipidemia indexes. Methods A cross-sectional health survey was carried out in three hospitals in Chongqing.The random samples came from 365 males in physical examination centers;among them 242 patients with high level of blood lipids were used as case group;and the other 114 healthy subjects were used as control group.The plasma levels of total cholesterol (TC),triglycerides(TG),high-density lipoprotein cholesterol (HDL-C),low-density lipoprotein cholesterol(LDL-C) were determined;the heights and body weights were detected.The genotypes of APOE were evaluated by gene chip sequencing method. Results Six kinds of genotypes in the population were find,among them E3/3 had the highest frequency of APOE genotypes (69.10%),meanwhile the frequency of APOE E2 allele was 17.93%.The frequency of E3 was 75.84%,and the frequency of E4 was 6.18%.There were statistically significant differences of the TC levels between the patients with different APOE genotypes in case group (P<0.05),meanwhile the E4 carriers had a higher TC level than E2 and E3 carriers (P<0.05).The multiple linear regression analysis showed that APOE polymorphism was positively related with the levels of TC and TG(r=0.157,P<0.05;r=0.276,P<0.05),however it was negatively related with the HDL level(r=-0.153,P<0.05). Conclusion The polymorphism of APOE is related to the blood lipid levels in Chinese male population,while the patients with APOE4 genotype have higher TC and TG levels.

Objective To explore the expressions of matrix metalloproteinase-2(MMP-2) and matrix metalloproteinase-9(MMP-9) in the tumor tissue of the patients with esophageal squamous cell carcinoma, and to analyze the correlation between the expressions of MMP-2 and MMP-9. Methods 100 spicemens of surgical removed esophageal squamous cell carcinoma were chosen. The tumor tissue and the adjacent normal esophagel mucosa tissue were selected.RT-PCR method was used to detect the expressions of MMP-2 and MMP-9 mRNA in the esophageal squamous cell carcinoma tissue and the adjacent normal tissue.Western blotting method was used to testify the expressions of MMP-2 and MMP-9 proteins;the correlation between the expressions of MMP-2 and MMP-9 was analyzed by Pearson correlation analysis. Results The positive expression rate of MMP-2 mRNA in carcinoma tissue was 89.0%(89/100), the positive expression rate of MMP-2 mRNA in adjacent normal tissue was 76.0%(76/100),and there was statistical difference between two groups(χ²=5.853,P=0.016).The positive expression rate of MMP-9 mRNA in carcinoma tissue was 48.0%(48/100),the positive expression rate in adjacent normal tissue was 16.0%(16/100),and there was statistical difference of the positive expression rate between two groups(χ²=23.529,P=0.000).There was a positive correlation between the expression of MMP-2 mRNA and the tumor invasion depth(χ²=4.971,P=0.026), but there was no correlation between the expression of MMP-2 mRNA and the lymph node metastasis(χ²=2.127,P=0.145).There was a positive correlation between the expression of MMP-9 mRNA and the tumor invasion depth(χ²=4.421,P=0.035), but there was no correlation between the expression of MMP-9 mRNA and lymph node metastasis(χ²=0.617,P=0.432). There was positive correlationship between the positive expression rates of MMP-2 mRNA and MMP-9 mRNA in esophageal cancer tissue(r=0.274,P=0.006). Conclusion The MMP-2 and MMP-9 may play a very important role in the occurrence and development of esophageal squamous cell carcinoma,however they have no affection on lymph node metastasis of esophageal squamous cell carcinoma.

Objective To investigate the relationship between the second infection and the mortality of the patients with cirrhosis, and to elucidate the influence of the second infection in the risk of death of the patients with cirrhosis. Methods A total of 103 patients with cirrhosis were enrolled and divided into first infection group (n=78) and second infection group (n=25).The basic characteristics,model for end-stage liver disease (MELD) scores,serum albumin levels,microorganisms and mortality rates in 30 d as well as the incidence of infection in parts of body of the patients in two groups were compared,and the risk factors for death in the patients with cirrhosis were analyzed by multivariate Logistic regression model. Results The mortality of the patients in second infection group was significantly higher than that in first infection group (P<0.05).The incidence of pneumonia and clostridium difficile-associated diarrhea (CDAD) of the patients in second infection group was significantly increased compared with first infection group (P<0.05).Logistic regression analysis illustrated that the higher MELD scores (OR=1.293,P<0.0001),existence of second infection (OR=4.582,P=0.0002) and lower serum albumin level (OR=0.487,P=0.0387) were the independent risk factors for the death in patients with cirrhosis. Conclusion Second infection independently increase the risk of death of the hospitalized patients with cirrhosis.

Objective To explore the association between single nucleotide polymorphisms(SNPs) of angiotensin converting enzyme (ACE) and susceptibility of coronary heart disease in Han population from North China,and to clarify whether ACE gene is the susceptible gene of coronary heart disease. Methods Using case-control study,246 patients with coronary heart disease confirmed with coronary angiography were used as case group,and 251 health persons were selected as control group.PCR-RFLP was applied to detect the SNPs of rs4267385 and rs4316 in ACE gene. Statistical software SPSS17.0 was used to analyze the assiation between ACE gene polymorphisms and coronary heart disease. Results The genotype distributions of the two SNPs(rs4267385,rs4316) didn't deviate from Hardy-Weinberg equilibrium in both case and control groups(P> 0.05); the allelic and genotypic frequency distributions of rs4267385 in ACE gene showed significant differences betwwen case and control groups(P<0.05),and the allelic and genotypic frequency distributions of rs4316 in ACE gene showed no significant differences betwwen case and control groups(P> 0.05). Conclusion ACE gene is likely to be the susceptible gene of coronary heart disease in Han population from North China.

Objective To investigate the relationship between the expressions of S100 calcium-binding protein P (S100P) in gastric cancer tissue and serum of the patients with gastric cancer and their clinicopathological parameters and monitor the serum S100P protein levels before and after tumor resection,and to explore its correlation with the prognosis of gastric cancer. Methods 50 patients with gastric cancer confirmed by surgical resection and pathological examination were selected.The pre-operative serum and post-operative gastric cancer tissue paraffin samples were collected.The fresh tissue samples and adjacent tissue were adopted from 20 patients with gastric cancer,and the postoperative dynamic serum samples were obtained from the other 30 patients with gastric cancer.Meanwhile,30 health volunteers were selected as normal control group.The expression level of S100P mRNA in gastric cancer tissue was detected by semi-quantitative RT-PCR.Immunohistochemistry and enzyme linked immunosorbent assay (ELISA) were used to detect the expression levels of S100P protein both in gastric cancer tissue and serum. Results The expression level of S100P mRNA in gastric cancer tissue was significantly lower than that in adjacent normal tissue (P<0.05);the positive expression rate of S100P protein in gastric cancer tissue was significantly lower than that in adjacent tissue (P<0.05);the down-regulation of the expression of S100P protein in gastric cancer tissue was positively correlated with vascular cancer embolus (P<0.05).The serum S100P level in the patients with gastric cancer before operation was lower than that in normal control group,but there was no significant difference (P> 0.05).The serum S100P level in the gastric cancer patients without lymph node metastasis was higher than that in the patients with lymph node metastasis,but there was no significant difference (P> 0.05).The serum S100P level in the patients with gastric cancer 2 weeks after operation was significantly lower than before operation. Conclusion The expression levels of S100P in gastric cancer tissue and serum of the patients with gastric cancer are decreased,and its expression level is correlated with the prognosis. The continuous dynamic monitoring of serum S100P level may predict the recurrence and prognosis.

Objective To evaluate the clinical effect of the application of the new Quartz Splint^TM high strength silica fiber splint in keeping the orthodontic teeth,and to provide the basis for choosing appropriate orthodontic retainer. Methods A total of 62 patients needing local strengthening retainer after completed orthodontic treatment were selected.The patients were divided 3-month,6-month and 12-monthgroups according to the follow-up time.Quartz Splint^TM high strength silica fiber splint was used to fox the labial teeth.Visual and instrument examination were used to check wether there were off-sites of retainers in various groups,teeth gap and the presence of recurrent dislocation;the periodontal health status was observed including probing depth(PD),bleeding index(BI),and plaque index(PLI). Results 60 patients using Quartz Splint^TM high strength silica fiber splint were stable without frature while the periodontal tissue revealed no significant changes in all 62 patients.Only 2 cases of Quartz Splint^TM high strength silica fiber splint in 6 months and 12 months respectively had individual off-sites without breaking.During 12-month follow-up period,the total intact rate of Quartz Splint^TM high strength silica fiber splint was 96.77%,and the patients had healthy periodontal conditions. Conclusion The stable clinical effect can be obtained by the application of Quartz Splint^TM high strength silica fiber splint for scattered clearance of the front teeth area or rotated teeth after orthodontic.

Objective To explore the differences of clinical efficacy and safety between Argon helium cryoablation combined with intraveneous chemotherapy and intravenous chemotherapy,argon helium cryoablation in the treatment of advanced non small cell lung cancer(NSCLC). Methods The clinical materials of 89 patients with advanced NSCLC and failure in radiotherapy and (or) chemotherapy treated in Department of Interventional Radiology of Cancer Hospital of Tianjin Medical University were analyzed,and they were divided into chemotherapy alone group(n=26),cryoablation alone group(n=30),and cryoablation combined with chemotherapy group(combination group,n=33).The adverse reactions and complications after 2 months of treatment were analyzed.FACT-G scale was used to evaluate the life quality of the patients before and 2 months after treatment.After the treatment,every 1-2 months the follow-up with enhanced CT was performed,the objective response rate of the tumor was evaluated by modified response evaluation criteria in solid tumors (mRECIST).The progression-free survival and overall survival were analyzed with Kaplan-Meier method,and Log-rank test was used to identify the differences in the survival rate. Results Compared with chemotherapy alone group,the incidence rates of bone marrow suppression,gastrointestinal reaction,liver and kidney function damage of the patients in combination group had no significant differences (P>0.05).Two months after treatment,compared with before treatment, the total scores of life quality of the patients in three groups were improved in different degrees,and there were significant differences between before treatment and after treatment in combination group and cryoablation alone group(P<0.05),but there was no significant difference between before treatment and after treatment in chemotherapy alone group(P>0.05).Two months after treatment,the tumor response rates(CR+PR)in combination group and cryoablation alone group were higher than that in chemotherapy alone group,and there was no significant difference between combination group and cryoablation alone group(P>0.05).The median follow-up period was 17 months,the median progression free survival of the patients in three groups were 11.1 months in combination group,6.9 months in cryoablation alone group,and 6.1 months in chemotherapy alone group;there were significant differences between combination group and other two groups(P<0.05).The median survival of the patients in combination group,cryoablation alone group and chemotherapy alone group were 16 months,13 months and 10 months;there were significant differences between combination group and other two groups(P<0.05). Conclusion Cryoablation combined with chemotherapy is a safe and effective method in the treatment of the patients with advanced NSCLC after failure in radiotherapy and (or) chemotherapy,and its curative effect is better than intravenous chemotherapy and cryoablation alone.

Objective To analyze the electrophysiological changes of palmar cutaneous brahch of median nerve (PCBm) in the patients with carpal tunnel syndrome(CTS) by detecting the electromyogram of PCBm and routine nerve conduction studies (NCS) of the CTS patients. Methods 50 patients who were diagnosed as CTS according to clinical features and electromyogram were used as CTS group and 30 cases of healthy persons were used as healthy control group.NCS were performed in all subjects.The complex muscle action potentials (CMAP) amplitude (Amp),distal latencies (Lat),motor conductive velocities (MCV),sensory nerve action potentials (SNAP) Amp,distal latencies (Lat)and sensory conductive velocities (SCV) were recorded,and the electromyogram of PCBm was performed. Results Compared with healthy control group, abnormal NCS of median nerve was found in all patients with CTS (100%),and the most common abnormal features were prolonged CMAP distal Lat and digits 3 Lat(P<0.01),and lower SCV of digits 3(P<0.01).29 (45.3%) patients had PCBm involvement,mainly exhibited as prolonged Lat(P<0.01), and lower SCV(P<0.01). Conclusion PCBm injury is commonly seen in the patients CTS,NCS of PCBm should be performed in the patients with CTS,and it should be considered as a definition that the compression of median nerve is beyond the carpal tunnel.

Objective To explore the advantages and disadvantages of the intensity modulated arc therapy(IMAT) plans and intensity modulated radiation therapy(IMRT) plan,and to provide the basis for their clinical application. Methods 19 patients with cervical cancer after operation were selected.The patients were scanned by simulation CT,and the targets and organs at risk were contoured. IMAT plan and IMRT plan were designed respectively in Eclipse 8.6 planning system.The treatment time and differences of the dose distribution in the targets and organs at risk of IMAT and IMRT plans were compared. Results The average design time of IMAT and IMRT plans of the 19 patients was (129±3) and (30±1) min(P=0.000);the average treatment time on Varian IX accelerator was (3.17±0.23) and (6.55±0.17) min(P=0.009).The homogeneity index(HI) of targets were 1.08 ± 0.01 and 1.10 ± 0.01(P=0.175).The conformal index(CI) of targets were 0.88 ± 0.01 and 0.82 ± 0.01(P=0.000).IMRT plan had the smaller doses at rectum,bladder,small intestine and neck of femoral compared with IMAT. Conclusion Compared with IMRT,IMAT plan has the advantages in dose distribution at targets and CI,its HI and doses of organs at risk are almost same with IMRT.Compared with IMRT,the design time of IMRT plan is increased three times,and the treatment time is reduced by half.In clinic,if the planning time is affluent,the IMAT plan is recommended.

Objective To investigate the clinical features of reflex anuria after radical hysterectomy,and to analyze its diagnosis and treatment methods. Methods Combined with reviewing the relevant literature,the clinical data of a case of reflex anuria after radical hysterectomy in our hospital was retrospectively analyzed.This patient developed anuria of 69 h duration after radical hysterectomy in our hospital,and the serum creatinine level gradlually increased to 620.7 μmol·L^-1.The patient was managed with correction of fluid and electrolyte imbalances,diuretics,and relieving the intrarenal arteriolar spasm and ureteral spasm.At the same time,the patient underwent ureteral catheterization and control of hypertension. Results By the 69th hour after undergoing radical hysterectomy in the patient,diuresis was established.The kidney function continued to improve for the remaining course of the hospitalization.The micturition and the kidney function of the patients were normal during the 4 months following-up. Conclusion Reflex anuria which is proved to cause acute kidney injury is rare in clinic,and it is easily misdiagnosed.The patients with reflex anuria can be successfully managed with medical or surgical interventions if this disease can be early diagnosis.

Objective To investigate the effects of extraction time,concentration of acetic acid and solid-liquid ratio on the extraction of rat tail tendon collagen,and to establish and optimize the extraction process of rat tail tendon collagen,and to increase the extraction efficiency. Methods The healthy SD rats tail were selected,the rat tail tendon collagen was extracted by acid-soluble method,the influence of different extraction time (12,24,48,72,96 h),acetic acid concentrations (0.10,0.25,0.50,0.75,1 mol·L^-1) and solid-liquid ratios (1:5,1:10,1:20,1:40,1:80) in the extraction ratio of collagen was observed. The optimal extraction parameters were explored by orthogonal test to judge the extraction time,the concentration of acetic acid and solid-liquid ratio,etc. The extracted collagen was identified by SDS-PAGE electrophoresis,tannic acid precipitation test and Fourier transform infrared spectroscopy test etc. Results The univariate analysis results showed that the collagen extraction rates were increased with the increasing of the extraction time,the concentration of acetic acid and the liquid ratio within a certain range of parameters. The optimal parameters were the extraction time of 72 h,the concentration of acetic acid of 0.5 mol·L^-1,the solid-liquid ratio of 1:40.SDS-PAGE electrophoresis showed that the samples were mainly collagen typeⅠ.Tannic acid precipitation test showed a low degree of degradation of collagen.The extract was confirmed by infrared spectral analysis to be typeⅠcollagen with intact structure.After cross-linking of collagen,the SEM figure showed that the internal interconnection formed a porous honeycomb structure,and the pore size was suitable. Conclusion The extraction process of rat tail tendon collagen is successfully established and optimized.The optimum process parameters of the extraction time,the acetic acid concentration and the material liquid ratio are gained,which can improve the extraction efficiency of rat tail tendon collagen.

Objective To prepare the polyclonal antibody against non-structural NP1 of minute virus of canine (MVC), and to identify its properties. Methods The target NP1 gene of MVC was amplified from MVC infection clone (pI-MVC),and was inserted into the pGEX-4T-3 vector to form the recombinant plasmid. Then the prokaryotic expression vector of pGEX-4T-3-NP1 was transform into E.coli (BL21),and the GST-NP1 fusion protein was induced under the optimized induction of isopropyl β-D-1-thiogalactopyranoside(IPTG).The recombinant proteins were purified using affinity chromatography.The purified fusion protein was inoculated into the adult rabbits to develop antiserum.After the titer of the antiserum was detected by ELISA,Western blotting and immunofluorescence methods were performed to evaluate the features of the prepared antiserum. Results The prokaryotic expression vector pGEX-4T-3-NP1 of MVC NP1 gene was successfully constructed.The soluble recombinant protein was highly expressed in E.coli BL21,and then it was purified and inoculated into the adult rabbits to obtain high titer antiserum.The ELISA result showed that the titer of the antiserum was 1:400 000.The Western blotting and immunofluorescence results showed that the specificity of the prepared antiserum was perfect. Conclusion The antiserum of NP1 prepared from MVC GST-NP1 fusion protein has shown a high titer and high specificity against NP1 proteins,which can be used for further study of NP1 in the molecular mechanisms of MVC infection.