Objective To construct the recombinant vectors that coexpress red fluorescent protein and small hairpin RNA (shRNA). Methods The subcloning, T-A cloning, and PCR technique were used to construct the recombinant vectors,named pcDNA3.0/DsRed-U6-shGFP and pcDNA3.0/DsRed-U6. Results The recombinant expression vectors mentioned above were successfully constructed.Conclusion The successful construction of recombinant vectors can establish a feasibility to further explore the functions of specific genes in eukaryotic cells with RNAi technique

Objective To study the VEGF-induced expressions of Ets-1 of human umbilical vein endothelial cells(HUVECs) cultured in vitro. Methods The HUVECs were cultured and obtained. The immunocytochemical stain and Image-Pro Plus (IPP) analysis system were used to determine the VEGF-induced expressions of Ets-1 in the cultured endothelial cells. The relative indexes were analyzed with semi-quantitative technique. Results The VEGF (10 μg•L^-1) induced the short-term expressions of Ets-1 in HUVECs (P<0.01), and Ets-1 expressions increased with the prolongation of time. The expressions of Ets-1 protein in HUVECs reached a peak at 10 min. After 30 min, the intensity of the expressions of Ets-1 decreased gradually to the level of no VEGF-induced. Conclusion The VEGF can induce the short-term expressions increase of Ets-1 of HUVECs.

Objective To construct the radiation-inducible expression vector pEgr-hTRAIL containing human TNF related apoptosis inducing ligand (hTRAIL) gene and study its expression and function of inducing apoptosis in A549 human lung adenocarcinoma cells. Methods Expression vector pEgr-hTRAIL was constructed with DNA recombinant technique. pEgr-hTRAIL plasmids were packaged with lipofectin to transfect into A549 cells in vitro. Stably transfected cell line A549-shTRAIL was selected through G418. The expression of hTRAIL gene was detected by RT-PCR. The early stage apoptosis of A549 cells was detected by Annexin-V-FITC apoptosis detecting kit. Results Expression vector pEgr- hTRAIL was constructed correctly by identification with restriction enzyme digestion. The expression of hTRAIL mRNA in stably transfected cell line A549-shTRAIL was increased significantly. The percentage of apoptotic A549-shTRAIL was increased significantly; it was 1.8 times as much as A549 cells (P<0.05). Conclusion Expression vector pEgr-hTRAIL is constructed successfully,which can increase the apoptosis of the stably transfected cell line A549-shTRAIL significantly.

Objective To study the role of basolateral amygdala (BLA) in startle conditioned reflex. Methods The rats were divided into two groups. The responses of neuronal discharges of BLA to the foot shock and the tone stimulus by glass microelectrodes were observed and recorded in acutely experimental rats. The cannulation of femoral artery in chronically experimental rats was performed. The startle conditioned reflex was started after 3 days. The training was to combine a conditioned stimulus (a tone) with an unconditioned stimulus (a foot shock), the startle responses were established by only conditioned stimulus in chronically experimental rats. After the roles of BLA were blocked by 0.3 μL 2% lidocaine, the response to conditioned stimulus was observed. Results The neuronal discharges of BLA were increased after the tone stimulus or the foot shock (change of discharges frequency>20％) in the acutely experimental rats. The blood pressure was not increased before training （P＞0.05）, but the blood pressure was increased after 3 days training (P＜0.01) by only tone stimulus in the chronically experimental rats. The blood pressure was not increased by only tone stimulus (P＞0.05) when the BLA was blocked by lidocaine. Conclusion The BLA neurons can experience the tone and the foot shock and play an important role in startle conditioned reflex.

Objective To investigate of the mutations in the PreC G1896A and in the basic core promoter (BCP) A1762T and G1764A as well as the effects of YMDD mutants on Lamivudine therapy against chronic hepatitis B virus (HBV) infection.Methods Serum samples were obtained before, during and after therapy. Parts of the HBV polymerase gene flanking the YMDD motif and the PreC/BCP region were sequenced after polymerase chain reaction (PCR) amplification. Results ①There were no significant differences of the mutations in PreC G1896A and BCP A1762T,G1764A between patients with viral breakthrough (VBT) and without VBT, as well as patients with seroconversion and without seroconversion (P>0.05).②There were significant difference of mutations in G1896A between patients with and without YMDD mutants (P<0.05), and the mutation detection rate (9%) in patients with YMDD mutants was lower. ③There was significant difference of YMDD mutants between patients with VBT and without VBT (P<0.05). Conclusion The mutations in PreC G1896A and BCP A1762T, G1764A have not effects on VBT and seroconversion following Lamivudine therapy,and G1896A may reduce the appearance of YMDD. The YMDD mutants may be one of the factors that aggravate the damage of hepatocytes during the relapse.

Objective To investigate the anti-oxidative stress effects of Taurine on rat hepatic stellate cells (HSC) cultured in vitro. Methods The freshly isolated rat HSC was incubated with general culture mediums and mediums with 50 or 25 mmol•L^-1 of Taurine,respectively, the levels of LPO and hydroxyproline in supernatant as well as the expressions of TGF-β1 by HSC in different mediums were evaluated. The effect of Taurine on proliferation of HSC was verified by BrdU method. Results With the activation of HSC, LPO and hydroxyproline expressed into three mediums were increased significantly（P<0.05）.This up-regulation was suppressed significantly by administration of 50 mmol•L^-1 or 25 mmol•L^-1 Taurine (P<0.05); Meanwhile, compared with the general medium group, the expressions of TGF-β 1 mRNA were decreased dramatically in HSC incubated with 50 mmol•L^-1 or 25 mmol•L^-1 Taurine pre-treated mediums（P<0.05）; furthermore, administration of 50 mmol•L^-1 Taurine suppressed the proliferation of HSC significantly（P<0.05）. Conclusion Taurine is effective in inhibiting the production of LPO as well as hydroxyproline by HSC and inhibiting proliferation of HSC, suggesting that Taurine, as an anti-oxidative stress factor, may have a potential therapeutic value on liver fibrosis.

Objective To explore the activation and maturation of dendritic cells (DC) stimulated by immune complex. Methods Immune complex was added to the immature DC induced by IL-4 and GM-CSF from PBMC and the expressions of costimulatory molecules CD86 on the surface of DC and cytokines in the supernatant were detected. Results Compared with antigen or antibody, immune complex could significantly activate the DC by upregulating the expression of CD86 on the surface of DC which was the second signal for the activation of cytotoxic T lymphocytes and the secretions of IL-6 and TNFα. Conclusion Immune complex can induce the activation and maturation of DC.

Objective To clone the human CD200 gene and to construct the expression plasmid pcDNA3-CD200. Methods The CD200 gene was obtained with the technique of RT-PCR from human peripheral white cells at 62 h after stimulation with 200 μg Con A.With automatic sequencing of pMD18T-CD200, the pcDNA3-CD200 recombinant plasmid was constructed by subclone, CD200 gene was obtained from human peripheral white cell. Results The obtained CD200 gene was completely consistent with the human CD200 sequence in GenBank (NM-005944). Conclusion The human CD200 gene is successfully cloned and pcDNA3-CD200 recombinant plasmid is constructed.

Objective To study whether lateral habenular nucleus (LHb) was involved in the central decrease of blood pressure induced by melatonin (MEL). Methods The neuron spontaneous firings were recorded by a single-barrel glass electrode and the excitatory or inhibitory cardiovascular neurons were identified by recording the changes of neuron firing sequences induced by intravenous injection with noradrenaline. After rats were administrated with MEL or physiological saline (ip or microinjection into LHb), the firings of cardiovascular neurons and artery blood pressure were recorded. Results The administration of MEL (2 mg•kg^-1, ip) could significantly vary the spontaneous firing sequences of cardiovascular neurons, leading to the decrease of excitatory neurons (38.40%±6.48%) and the increase of inhibitory neurons (56.30%±8.45%), compared with physiological saline control. The microinjection of MEL (4 mg•kg^-1) into LHb also induced the decrease of artery blood pressure (1.57 kPa±0.35 kPa). Conclusion MEL could act on LHb and result in the decrease of artery blood pressure. So LHb could be the central site involved in the decrease of blood pressure induced by MEL.

Objective To observe the effects of lysophosphatidic acid (LPA) on voltage dependent potassium current in myocytes of guinea pig. Methods The whole cell patch clamp technique was used in isolated ventricular cells of guinea pigs. Results Thirty-three voltage dependent potassium current in myocytes were recorded. LPA (1.0 and 10.0 μmol•L^-1) could significantly decrease the amplitude of the potassium current of ventricular myocytes. Conclusion LPA may participate in arrhythmia occurrence during myocardial infarction.

Objective To investigate the relationship between F11R gene polymorphism on chromosome 1 and schizophrenia in Chinese population. Methods The PCR-RFLP method was conducted to examine the genotypes of F11R genes in 242 subjects including 128 patients with schizophrenia (case group) and 114 healthy controls (control group), and then the frequencies of genotype and allele of F11R gene were statistically computed. Results There were no remarkable differences of the frequency distributions of A and G of F11R gene between cases and controls （χ²=0.498， P>0.05）, and the frequency distributions of AA and AG had no markedly differences between cases and controls （χ²=0.533， P>0.05）. Conclusion The F11R gene on chromosome 1 may be not the susceptible gene of schizophrenia.

Objective To study the influences of intense infrasound on the ultrastructural changes of the hair cells of cochlea in guinea pigs with electron microscope. Methods Thirty guinea pigs were randomly divided into five groups: control group and 4 experimental groups. The guinea pigs in experimental groups were individually exposed to 8 Hz, 12 Hz, 120 dB SPL, and 130 dB SPL in a reverberant chamber. The animals were scarified for 5 h, once a day for 4 consecutive days. The influences of intense infrasound on the ultrastructural changes of hair cells of cochlea in guinea pigs were observed by transmission electron microscope. Results The hair cells of cochlea in all experimental groups were damaged to different extent including mitochondrion denaturation, nucleus expansion, and karyopyknosis. The outer hair cells were damaged remarkably, but inner hair cells were nomal; the hair cells in 130 dB SPL group were significantly damaged compared with 120 dB SPL group. There was no difference of damage of hair cells between 12 Hz group and 8 Hz group. Conclusion The intense infrasound can cause damages with different extent to the ultrastructures of hair cells of cochlea in guinea pigs.

Objective To investigate the expressions of MMP-9 protein and MMP-9 mRNA in the tissues and cells of the gastric carcinoma and the effects of MMP-9 on the cancer progress. Methods The expressions of MMP-9 and MMP-9 mRNA in 74 cases and the BCG823 cells of gastric carcinoma cultured in vitro were examined by immunohistochemistry, in situ hybridzation, invasion test and morphometric analysis. The relationships between the expressions and the clinical parameters of gastric carcinoma were analysed. Results The expressions of MMP-9 and MMP-9 mRNA were closely related to the invasion, differentiation, and lymph nodes status of the gastric carcinoma. The MMP-9 and MMP-9 mRNA were positive in BCG823 cells. The antibody to MMP-9 could decrease the invasion and metastasis ability of tumor cells. Conclusion Gastric carcinoma cells have the ability to produce and secrete MMP-9 which can promote the invasion and metastasis of the neoplasm.

Objective To establish a method to isolate and identify human mensenchymal stem cells(HMSC). Methods The HMSC were isolated and cultured in vitro by applying methods of density-grad centrifugal and anchoring screening. The immunocytochemistry, cytochemistry, and electron microscopy were used to identify HMSC cultured in vitro. Results The HMSC have particular phenotype:CD29 and CD44 were positive,CD34 and CD45 were negative.PAS was positive,and SB was negative.HMSC were infantile under electron microscope. Conclusion The HMSC can be isolated with density-grad centrifugal and anchoring screening. The method to identify HMSC with immunocytochemistry, cytochemistry and electron microscopy is effective.

Objective To construct the pronucleus expression vector of human p21^waf1and express it in E.coli strain of JM109. Methods The total RNA was isolated from human hepatocytes, and p21^waf1 was cloned by RT-PCR, the PCR product was ligated with PGEM-T vector and sequenced. Then the target sequence was inserted into an temperature-sensitive expression vector pBV220 and expressed in E.coli JM109.The recombinant protein was identified by immunoblot. Results DNA sequencing confirmed that the sequence of cloned p21^waf1 was identical to the published sequence. The recombinant plasmid pBV220/p21^waf1 was transformed into E.coli JM109 and over- expressed during 42℃ induction. After induction for 5 h, the expression products showed a high expression level of recombinant protein with 21 000, and was more than 38% of the total bacterial protein. Western blotting analysis indicated that the the recombinant protein could react specifically with anti-P21^waf1 antibody. Conclusion The p21^waf1 expression vector is constructed successfully, and the induced-protein is confirmed as P21^waf1 protein by detection.

Objective To investigate the long-term effects of portal vein hemodynamic changes on liver function after portal vein arterialization(PVA) in rats. Methods The rats were divided into PVA group and control group. The common liver artery was anastomosed with the proximal portal vein by end-side way, and the distal portal vein with the vena cava by side-side way in PVA group for the sake of establishing the models of rat PVA. The abdominal cavity was closed after dissociating the liver hilar tissue in control group.The blood flow volume and cross section in two groups operated for one month and six months ,respectively, were observed. The portal vein pressure, liver function and structure after six months were observed. Results Compared with control group, the cross section and blood flow volume of portal vein had a increasing trend in PVA group（P<0.05）.Portal vein pressure,the levels of plasma endotoxin,serum albumin,bile acid salt,DBIL and TBIL didn′t present distinct difference（P>0.05）. But the level of serum GPT in PVA group increased evidently（P<0.05）,and the walls thickened and the proportion of collagen increased in distended portal vein and its branches. Conclusion The PVA without limiting the blood flow volume can increase the cross section and blood flow volume of portal vein ,dilate and thicken the branches of portal vein, but the function of reticuloendothelial system, metabolism of bile pigment and bile acid salt don′t undergo significant influences.

Objective To investigate the effects of high-voltage prickle electrostatic fields (HVPEF) on proliferation and drug susceptibility of mouse Lewis lung cancer cells. Methods MTT method was adopted to detect A values of cells for deducing relative activity of cells in group after HVPEF, group treated only with carboplatin, group treated with both HVPEF and carboplatin. Results In group after HVPEF, the cell growth was stimulated with voltages ≤1.5 kV. At voltage 1.5 kV， the relative activity of cells was 132.7% compared with control group(P<0.05); the cell growth was inhibited after voltages >1.5 kV, the best inhibitory voltage was 2.5 kV and the relative activity of cells in 2.5 kV group merely was 71.7% of that in control group (P<0.05); Moreover, the effects of HVPEF and carboplatin together on the cells were significant, the relative activity of cells in group treated with both HVPEF and carboplatin was 57.8% of that in control group (P<0.01). Conclusion Different intensities of HVPEF can directly inhibit the growth of tumor cells and increase the susceptibilities of tumor cells to chemotherapy drugs.

Objective To determine the role of Tat in mediating HIV encephalitis (HIVE). Methods The contents of tat mRNA in tissue extracts of 9 AIDS patients with HIVE and 7 AIDS patients without HIVE were measured with RT-PCR. Results Despite long autopsy times and significant degradation of RNA extracted from brain tissue, tat mRNA was detected in 4/9 patients with HIVE but not in any of the 7 patients without HIVE. Similarly, the env mRNA was also detected in 5/9 patients with HIVE but not in the patients without HIVE. However, vif mRNA was detected in both groups of patients with HIVE (5/9) or without HIVE (2/7). Conclusion This result suggests that Tat likely plays an important role in the neuropathogenesis of HIVE.

Objective To study the gene expression of astrocytes as the gene target cells. Methods The NGF, BDNF, and NT3 genes of rats were cloned, the eukaryote expression vectors were established, the three kind of recombinant vectors were used to transfect astrocytes, the positive cloned cells were cultured dilatedly after G418 sifting; using supernates of culture liquid of astrocytes modified by gene to culture PC12 or TrkB-PC12, the expression and its level of gene target cells aimed genes were measured by Western blotting or immunohistochemical method. Results After G418 sifting, astrocytes modified by gene transfected by NGF, BDNF, and NT3 gene showed G418 positive; PC12 cultured in supernate culture liquid transfected by NGF and NT3 showed increases in the number and longer proptosis, TrkB-PC12 cultured in supernate culture liquid transfected by BDNF also showed increases in the number and longer proptosis; largely G418 positive gene target cells showed immune staining positive. Western blotting showed a protein belt with corresponding molecular weight. Conclusion Astrocytes can effectually express and secrete NGF, BDNF, and NT3 with biological activties as gene target cells.

Objective To explore the molecular mechanism of protective effects of Panaxadiol Saponins (PDS) on lung tissues of endotoxic shock rats. Methods Wistar rats were randomly divided into control group (CTRG), lipopolysaccharide (LPS) shock group (LPSG), LPS shock+dexamethasone group (DEXG) and LPS shock+PDS group (PDSG), respectively. Endotoxin shock models were duplicated by injection of LPS (4 mg•kg^-1). The histological changes of the lung tissues stained with hematoxylin and eosin (HE) among four groups were compared through microscopic examination. Meanwhile, the expressions of CD14 and IκBα mRNA were measured by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and the expressions of CD14 and NF-κB proteins were determined by Western blotting assay. Results ①The histological and pathological changes: in LPSG, there was the effusion of a large amount of lymphocytes, mono-macrophages and neutrophils around the alveolar and vascular walls; incrassated alveolar septums and bronchiole walls were caused by hyperemia ,edema and infiltration of pulmonary interstitial;focal atrophia and formation of pneumatocele could be observed; only small amount of alveolus containing air could be found; however, the lung pathologic changes in DEXG and PDSG were significantly slighter than those in LPSG, and they merely showed slight effusion of inflammatory cells, and the content of air in alveolus was near to that in CTRG. ②The expressions of CD14 mRNA and protein in LPSG were significantly higher than those in CTRG（P<0.05）, while those in DEXG and PDSG were close to those in CTRG（P>0.05）, significantly lower than those in LPSG（P<0.05）. The expression of lung IκBα mRNA in LPSG was significantly lower than those in CTRG（P<0.01）, while those in DEXG and PDSG were close to that in CTRG（P>0.05） and higher than that in LPSG（P<0.05 and P<0.01）. The expression of NF-κB P65 protein in LPSG was significantly strengthened than that in CTRG（P<0.05）, while those in PDSG and DEXG were lower than that in LPSG（P<0.05）, close to that in CTRG（P>0.05）. Conclusion LPS activates the signaling pathway mediated by LPS receptors, the decreasing of IκBα mRNA expression and the increasing of CD14 and NF-kB expressions are the molecular mechanism for the lung tissue injuries, while the opposite changes in PDSG reveal the molecular mechanism of their protective effects on cells and tissues; PDS has the same effects with DEX to protect against the lung tissue injuries of endotoxic shock in rats.

Objective To clone mouse secretable Hemopexin（sPEX） cDNA, construct pEgr-sPEX recombinant plasmid and detect the expression of recombinant plasmid in B16F10 cells. Methods Hemopexin cDNA was amplified from the NIH3T3 cells by RT-PCR. After the cDNA identified by sequencing, the pEgr-sPEX recombinant plasmid was constructed and the plasmid was transfected into B16F10 cells with liposome and the expression of PEX induced by ionizing radiation in B16F10 cells was detected by Western blotting. Results The sequencing results proved the cloned sPEX cDNA to be completely identical with that reported in the GenBank. The mouse sPEX cDNA was inserted correctly into expression vector and expressed successfully. Conclusion The mouse sPEX cDNA is cloned successfully and it is confirmed that pEgr-sPEX possesses the radiation inducing expression characteristics in vitro.

Objective To delivery gene by electropermeabilization to skeletal muscles and choose the optimal parameters. Methods The enhanced green fluorescent proteins (EGFP) gene-encoding plasmid was used as reporter gene to investigate the conditions of best gene transfection with minimal muscle injury. Changing one parameter with other parameters fixed， transfection rates were compared，and optimal parameters to target plasmid DNA into skeletal muscles of mice were chosen. Results After 15 μg EGFP plasmid was injected into skeletal muscles of mice，8 square-ware pulses were applied at amplitudes 200 V•cm^-1， with duration 20 ms and 1 Hz frequency，and the transfection rate was (73.2±7.2)%. Transfection efficiencies were same within 1 hour since it was stable.The optimal dosage of DNA was 15 μg. Maximum expression of EGFP fusion proteins was obstained 7 days after EGFP transfection. Conclusion DNA electrotransfection in muscle better than the only injection of EGFP plasmid into muscle.

Objective To study the effects of Aralosides (As) on cardiac function in rats with experimental heart failure. Methods Twenty-four experimental heart failure rat models induced by 1.5% pentobarbital sodium were researched by using hemodynamic method and then were devided into control group(salt solution, 0.9 mg•kg^-1), As group(As, 10 mg•kg^-1) and deslanoside group (deslanoside, 0.04 mg•kg^-1). Every group was controlled with the hemodynamic value 5 minutes after heart failure. Left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVEDP), maximal rising and falling rate of ventricular pressure(±dp/dt^max) were recorded with 4-channel polygraph to observe the the effects of salt solution, Aralosides and deslanoside on cardiac function in rats with experimental heart failure. Results In As group, the LVSP was increased from (10.8±1.0) kPa to (14.5±1.8)kPa (P<0.001)，+dp/dt^max was increased from (163±24)kPa•s^-1 to (295±66)kPa•s^-1 (P<0.001)，-dp/dt^max was decreased from (-140±31) kPa•s^-1 to (-218 ±60) kPa•s^-1 (P<0.01) and LVEDP was decreased from (1.46±0.24) kPa to (1.28±0.17) kPa (P<0.05) after intravenous administration of As. In deslanoside group, the LVSP was increased from (10.5±1.2) kPa to (12.9±1.6) kPa (P<0.01)，+dp/dt^max was increased from (144±26) kPa•s^-1 to (222±53) kPa•s^-1 (P<0.001)，-dp/dt^max was decreased from (-132±23) kPa•s^-1 to (-190±32)kPa•s^-1(P<0.001) ，and LVEDP was decreased from (1.31±0.19) kPa to (1.14±0.18)kPa (P<0.05) after intravenous adminstration of deslanoside. But the hemodynamic value did not change after intravenous administration of salt solution (P>0.05). Conclusion As can induce a positive inotropic action on heart failure myocardium, performing in a similar manner as deslanoside.

Objective To investigate the effects of β-amyloid on learning memory and hippocampus neuron ultrastructures in aging rats induced by D-galactose (D-gal). Methods Using the water labyrinth test, the rats with quickly reaction were selected and randomly divided into four groups（n=12）:control group，Aβ group，D-gal group， and D-gal+Aβ group. In the D-gal group and D-gal+Aβ group, each animal was injected intraperitoneally with the D-gal 50 mg•kg^-1•d^-1 for 6 weeks, the decrepitude animal model was established. In the control group and Aβ group, each animal was injected intraperitoneally with same quantity of physiology saline. At the 7th week, the rats in Aβ group and D-gal+Aβ group were injected with Aβ_25-35 into bilateral hippocampal regions, 1 μL in each side; and the rats in control group and D-gal group were injected with same quantity of physiology saline. After the model was established successfully, Morris water maza test was used to determine the ability of learning and memory. Ultrastructures of the hippocampus neurons were observed under electron microscope. Results ①One week after operation, the escape latencies were longer in Aβ group, D-gal group, and Aβ+D-gal group than that in control group (P<0.05),obviously in Aβ+D-gal group (P<0.01). ②The hippocampus neuron ultrastructures in control group were nearly normal, mitochondria cristae of the hippocampus neurons in Aβ group broke. The hippocampus neurons in D-gal group had lipofuscin deposit. In D-gal+Aβ group, nucleuses pyknosis was fonud and chondriosomes swelled. The crista broke and became emptey bubble. Conclusion The combination of D-gal and Aβ can induce a conspicuous reduction of learning-memory ability in rats, and increase the aging degree of brain.

Objective To investigate the inhibitory effects of Taurine on the cellular toxicity induced by glycolaldehyde. Methods The inhibitory effects of Taurine (50 mmol•L^-1) on glycolaldehyde (0.1 mmol•L^-1) induced DNA damage in spleen lymphocytes of mice were studied in vitro using single cell gel electrophoresis (comet assay)，and 2,4-dinitrophenylhydrazine (DNPH) measurement was used to detect quantitatively the effects of Taurine on carbonyl compound contents in a reactive system. Results After 50 mmol•L^-1 Taurine was added to the spleen lymphocyte suspension containing 0.1 mmol•L^-1 glycolaldehyde, the cellular DNA damage rates were markedly decreased (P<0.01) and the migration distances of DNA were significantly reduced (P<0.01) as compared with positive control group, and Taurine reduced the carbonyl compound contents in a reactive system in a dose-dependent manner. Conclusion Taurine can significantly inhibit the cellular toxicity induced by glycolaldehyde.

Objective Using skylight tumor model to observe the tumor growth and effects of angiostatin(AS) and endostatin(ES) on tumor. Methods The mice were randomly divided into four groups: blank contrast group, experimental contrast group, low dose experimental group(AS 0.6 ng•kg^-1＋ES：2.5 mg•kg^-1), and high dose experimental group(AS 1.2 ng•kg^-1＋ES 5.0 mg•kg^-1). Mice skylight tumor models were established.After 13 days, the tumor growth was prohibited obviously observed with the naked eye in high dose experimental group and the expressions of CD34 and VEGF were determined with immunohistochemistry to explore mechanism in the course of anti- angiogenesis. Results After the tumor cells had been transplanted into mice, it was difficult for the mice to go in experimental contrast group. But the tumor growth in drug experimental groups were slow and the mice could slow and walk well. The expressions of VEGF and CD34 were higher in experimental contrast group than those in drug experimental groups. Conclusion AS and ES can have important inhibitory effects on angiogenesis.

Objective To establish an effective method for purificating recombinant HspA-UreB fusion protein of Helicobacter pylori from inclusion body. Methods The inclusion bodies of HspA-UreB fusion proteins expressed in recombinant E.coli BL21（pET-HU27） were washed, denatured and renatured. The fusion proteins were isolated and purified by Ａ¨KTA Fast Protein Liquid Chromatogram（FPLC） with HiTrap Chelating HP, then the purity and content of target protein were detected by SDS-PAGE and Bradford method, and the immunocompetence was identified with Western blotting test. Results The recombinant HspA-UreB fusion protein from inclusion body was confirmed to have a purity and content of more than 90% and 0.25 g•L^-1, respectively, and a high immunocompetence. Conclusion An effective method for purificating recombinant HspA-UreB fusion protein of Helicobacter pylori from inclusion body is established successfully.

Objective To establish a method to isolate and culture the bone marrow-derived mesenchymal stem cells (BM-MSCs) in vitro and investigate their biologic characteristics. Methods The culture of mesenchymal stem cells was initiated from bone marrow low-density mononuclear cells separated by Pecoll centrifugation and maintained in low-glucose DMEM with 10% fetal calf serum. The cell growth pattern was evaluated by trypan blue exclusion. The cell cycle and surface antigenic features were analyzed by flow cytometry. Results The BM-MSCs were positive for CD29，CD44，CD166，HLA-ABC and negative for CD34, CD45,and HLA-DR. Cell cycle analysis showed that 12.8% of them was in S phase and 76.4% of them was in G₀/G₁ phase. The cells had a population doubling time of 2-3 days. Conclusion BM-MSCs are a homogenous monoclone population of cells with unique growth phenotype characteristics.

Objective To apply skin mesenchymal stem cells (MSCs) irradiated with different dosages of X-rays, instead of MSCs in the bone marrow to promote the proliferation of hematopoietic stem cells in vitro. Methods The bone marrow cells were cultured on the skin MSCs layer irradiated with different dosages of X-rays in vitro. The number of bone marrow cells, the colony-forming-unit-macrophage-granulocyte (CFU-GM) were assayed after expansion and maintenance for 0-12 days. CFU-GM, colony-forming unit-erthroid（CFU-E）, burst-forming-unit-erythroid （BFU-E）colony were assayed in the cultured supernatant of the skin MSCs. The effects of supporting hematopoiesis of irradiation on skin MSCs in fetal mice were observed. Results The CFU-GM colonies were increased after bone marrow cells were cultured together with fetal mice skin MSCs irradiated with 6,8, and 10 Gy X-rays, especially in 6 Gy X-rays group, it was 2.17 times as big as that in 0 Gy X-rays group. The cultured supernatant of skin MSCs irradiated with 6,8 and 10 Gy X-rays could stimulate the growth of CFU-GM, CFU-E, and BFU-E in vitro, there were significant differences compared with those in 0 Gy X-rays group (P<0.05), especially in 6 Gy X-rays group, they were 1.81,3.94,and 3.51 times as big as those in 0 Gy X-rays group,respectively.Conclusion Skin MSCs irradiated with certain dosage of X-rays in vitro have the function of sustaining the proliferation of hematopoietic cells,both irradiated MSCs layer and supernatant can increase the number of bone marrow cells colony.

Objective To observe the dynamic changes of MMP-2 and TIMP-2 after middle cerebral artery (MCA) ischemia in rats and evaluate the neuroprotective effects of Captopril on focal cerebral ischemia. Methods The rat focal cerebral ischemia models were made by using ligating the middle cerebral artery. The changes of MMP-2 and TIMP-2 in rat brain tissues in different groups were measured by immunohistochemical method. Results The MMP-2 expression was significantly incresed in the ischemia regions 5 d after inschemia (P<0.05), and Captopril could decrease the expression of MMP-2 (P<0.05); there were no differences of the effects of Captopril on TIMP-2 between various groups. Conclusion MMP-2 can aggravate cerebral damage after ischemia,and Captopril, an artificial inhibitor of metalloproteinase, plays an important protective role in brain.

Objective To investigate the effects of Cimetidine on portal hemodynamics in dogs with cirrhosis and portal hypertension. Methods Cirrhosis and portal hypertension were successfully induced in eleven dogs. These dogs were then divided into Cimetidine group (n=7) and saline control group (n=4),selected other 4 dogs as a normal group. Phisiological polygraph, electromagnetic flowmeters and other devices were used to detect the changes of portal and systemic hemodynamics in the normal group,saline control group, and Cimetidine group after portal hypertensive dogs in Cimetidine group were injected intravenously with Cimetidine (0.012 g•kg^-1 weight) and portal hypertensive dogs in salme control group with normal saline. Results In Cimetidine group，5，15，30，60，90, and 120 minutes after cimetidine injection, free portal pressure decreased 5.2% (P<0.05),14.1% (P<0.01), 13.7%(P<0.01),12.9%(P<0.01),9.3%（P<0.01）,6.5%(P<0.05), respectively. Wedged hepatic venous pressure, hepatic venous pressure gradient, portal venous resistance decreased synchronously. There was no significant change in hepatic arterial flow. Portal venous flow and total hepatic flow slightly increased. There were no significant changes in free hepatic venous pressure, inferior vena cava pressure, mean aorta arterial pressure and heart rate. The maximum decreased value of free portal pressure was significantly positively correlated to the histamine concentrations of femoral vein blood (r=0.787 4, P<0.05). No significant changes of any indexes mentioned above were found in saline control group after injection of normal saline and in normal group. Conclusion Cimetidine can reduce portal pressure, because it can reduce portal venous resistance by antagonizing the action of histamine. Cimetidine can slightly increase portal venous flow and has no bad effects on systemic hemodynamics.

Objective To study the proliferation of colony-forming units of granulocytes and macrophages (CFU-GM) and relationship with pathogenesis in BXSB lupus mice. Methods The formation of CFU-GM of bone marrow mononuclear cells (BMMC) of 4, 8, 12, 16 and 20-week-old male BXSB mice cultured in vitro with methylcellulose semisolid culture system was observed, the peripheral blood monocytes (PBMC) were counted and the urinary protein was test at the same time. C57BL mice with same ages were used as normal controls. Results The urinary protein emerged since 12 weeks in male BXSB, but remained negative in 4, 8-week-old male BXSB and C57BL with any ages. The CFU-GM was （96.00±18.11）/2×10⁵ BMMC, and peripheral blood monocytes reached （111.33±10.97）×10⁶•L^-1 in 8-week-old male BXSB mice, the counts were significantly higher compared with those of 4-week-old male BXSB mice and normal C57BL mice (P<0.05), and increased progressively in older BXSB mice. Conclusion The autoimmune disease of male BXSB mice may be caused by monocytosis which comes from the abnormal CFU-GM in the bone marrow.

Objective To establish the method of isolation,culture, and differentiation of Nestin positive cells of non-islet cells in adult rats in vitro. Methods The whole pancreas of three adult rats were digested in vitro with the intraductal collagenase distention method, then both islets and non-islets were collected and cultured with 10% fetal bovine serum (pH 7.6) and serum-free RPMI 1640 (pH 7.4).The formation process of new islet-like cell masses were examined by light microscopy,and the glucose-stimulated insulin release　(SI) from 40 just isolated islets and 40 new produced islet-like cell masses were tested.The expression of Nestin was tested by immunocytochemistry. Results Functioning islets in good morphology could be obtained from pancreas of adult rats by the intraductal collagenase distention method；there were adherent cells after 36 h culture,but no islets; the Nestin positive cells proliferated fast and formed new islet-like cell masses after 18-22 d. Nestin expression could be confirmed among adherent cells，but no insulin and glucagon, and Nestin positive cells can be seen in passage cells of new produced islet-like cell masses. Insulin secretion levels in 40 new isolated islets were (63.6±4.0)，(202.2±14.8)mIU•L^-1 in 3.3 and 16.7 mmol•L^-1 glucose, respectively;and (3.5±0.2)，(3.3±0.2)mIU•L^-1,respectively, in 40 new produced islet-like cell masses (P<0.001). Conclusion Functioning islets can be obtained from the pancreas of adult rats. Nestin positive cells of non-islet cells can be obtained from pancreas of adult rats in pH 7.6 condition through culture in vitro,and can form islet-like cell masses;Nestin positive cells of non-islet cells in pancreas of adult rats possess the characters of islet stem cells.

Objective To evaluate the functioin of a new muti-functional device of thoracic cavity closed drainage system with animal experimental study. Methods Twelve dogs were randomly divided into experimental and control groups (n=6). They were treated with two kinds of thoracic cavity closed drainage system respectively after artificial hemopneumothorax. The time of thoracic cavity closed drainage, anti-tensile strength,and hemoglobin contents in peripheral venous blood before operation and after reinfusion were observed. Results The time of thoracic cavity closed drainage in experimental group was (44.67±8.89)s and that in control group was (451.67±63.56)s, there was significantly difference (P<0.000 1). The anti-tensile strength of two devices in two groups were （55.17±0.71）N and （35.48±0.57）N (P<0.05)， respectively. The hemoglobin in experimental group was (106.33±9.56) g•L^-1 averagely after autotransfusion and that in control group was (63.83±7.19)g•L^-1 after transfusion with saline, there was significantly difference (P<0.001). All animals were survived in experimental group, whereas 3 animals were survived in control group in a period of 24 hours. Conclusion The new device is more availability and convenient than existing system. The new one has autotransfusion function specially.

Objective To study the effects of pleurodesis with the intrapleural injection of silver nitrate and talc in rabbits. Methods Intrapleural injection of 0.5% silver nitrate and talc slurry (400 mg•kg^-1), respectively, were performed to Japanese white rabbits to produce pleurodesis, the pleural effusion volume, white blood cell (WBC) numbers, the percentage of polymorphonuclear neutrophil (PMN%) and the content of lactate dehydrogenase (LDH) of 1 and 3 d after intrapleural injection were examined. The pleural adhesion scores of 1,3,7,14,28 d after pleurodesis in silver nitrate group and talc group were compared. Results The pleural effusion volume and WBC in silver nitrate group were higher than those in talc group at 1 and 3 d after pleurodesis (P<0.001), effusion volume and WBC at 3 d in the two groups were higher than those at 1 d (P<0.001); PMN% in both groups at 1 d were higher than those at 3 d (P<0.01), but it was lower at 3 d in silver nitrate group compared with talc group (P<0.001); the LDH contents in both groups at 1 d were higher than those at 3 d (P<0.001), and the LDH contents at 1 and 3 d in silver nitrate group were higher than those in talc group (P<0.01). The pleural adhesion score in silver nitrate group was higher than that in talc group (P<0.05). Conclusion Both silver nitrate and talc slurry can produce pleurodesis in rabbit pleura, and the low concentration of silver nitrate is superior to talc slurry.

Objective To study the pharmacological action of anti-inflammation, acesodyne, and anti-diarrhea of Fufangshanqikeliji (FFSQKLJ). Methods FFSQKLJ were poured consecutively into the stomach of healthy Wistar rats and Kunming mice, respectively. Then three kinds of models were set up 1 h after administration: inflammation models of the tumefaction of back metatarsus in rats by hypodermic injection (1% carrageenin 0.1 mL), ache models of twist in mice by celiac injection (0.7% glacial acetic acid 10 mL•kg^-1), diarrhea models in mice after pouring the senna. At the same time, control group (0.5% sodium carboxymethyl cellulose 20 mL•kg^-1) and salicylazosulfapyridine (SASP, 0.5 g•kg^-1•d^-1) group were set up. The tumefaction extents of back metatarsus of rats before and after inflammation, times of every mouse twist in 30 minutes after celiac injection (0.7% glacial acetic acid 10 mL•kg^-1), times of abnormal excretion in 1-6 h after pouring the senna, respectively, were observed. Results The tumefaction rates of back metatarsus in rats in the middle and large dosages of FFSQKLJ groups were much lower than that in control group (P<0.05 ), and the times of twist in mice were much lower than that in control group (P<0.05). Large, middle and small dosages of FFSQKLJ reduced the times of diarrhea in mice, compared with the control group, the differences were very obvious (P<0.05) . Conclusion FFSQKLJ have good effects of anti-inflammation, acesodyne, and anti-diarrhea.

Objective To evaluate the effects of panaxatriol in quinquefolium leaf (PQL) on immune functions in the patients with breast carcinoma undergoing radiotherapy. Methods Thirty patients with breast carcinoma undergoing radiotherapy were randomly divided into 2 groups: panaxatriol treatment group (n=17) and control group (n=13). All patients in panaxatriol treatment group were administrated with PQL as well as radiotherapy, no administration in control group. After radiotherapy with 40 Gy, the white cells count and IgG, IgA, IgM, C3, and C4 values, and the percentages of CD4⁺, CD8⁺, CD25⁺ and lymphocyte transformation rate of peripheral blood in the patients were examined. Results There were (4.02±0.67)×10⁹•L^-1 and (6.17±1.20)×10⁹•L^-1 white blood cells，respectively, in control and PQL groups. The lymphocyte transformation rate of peripheral blood were 7 966±1 562 in the control group, while 18 035±1 577 in PQL group, which was significantly higher than that in control group（P<0.001). The percentages of CD8⁺ and CD25⁺ T lymphocytes in PQL group were significantly higher than those in control group (P<0.05, P<0.001). Conclusion The PQL could enhance the cellular immune function in patients with breast carcinoma undergoing radiotherapy.

Objective To study the effects of CD4⁺ T lymphocytes on processing mononuclear phagocyte in patients with asthma. Methods The mononuclear phagocytes and CD4⁺ T lymphocytes were isolated from peripheral blood in 20 patients with asthma and 22 healthy donors, respectively. Every sample was divided into four groups:MΦA⁺，(MΦCD4⁺)A⁺，MΦ⁺CD4⁺A⁺，and MΦA⁺CD4⁺. All groups were stimulated with CTLL₂P antigen for 18 hours and CTLL₂P antigen were washed away, then they were cultured with B lymphocytes together, the levels of secreted CTLL₂P-specific IgM, IgG, and IgE in the supernatant were measured after 10 days. Results When CD4⁺ T lymphocytes were cultured with mononuclear phagocytes together, the level of CTLL₂ P-specific IgM of patients with asthma (A₄₉₀ nm)（0.033±0.031） was lower than that of healthy donors（0.098±0.062）(P<0.05); the level of CTLL₂ P-specific IgG of patients with asthma (A₄₉₀ nm)（0.080±0.035）was significantly higher than that of healthy donors（0.055±0.024）(P<0.05)；the same result was obstained (P<0.05), when primate CD4⁺ T lymphocytes were cultured with antigen processing mononuclear phagocytes (patients:0.075±0.018,healthy donors:0.057±0.018). The CTLL₂ P-specific IgE level of patients with asthma(0.154±0.119) was as same as healthy donors (0.186±0.101)(P>0.05). Conclusion The CD4⁺ T lymphocytes of patients with asthma have different regulatory functions on immunolglobulins from antigen processing mononuclear phagocytes.

Objective To explore the correlations of the changes of soluble Fas（sFas） levels with pathogenesis of breast diseases. Methods The serum sFas levels in 226 healthy women, 20 patients with recurrent breast cancer, 81 patients with primary breast cancer and 22 patients with benign breast diseases were detected by enzyme-link immunosorbent assay. Results In the healthy group,sFas level increased with age. The mean sFas levels in primary and recurrent cancer patients were 0.815 and 1.510 ng•L^-1，respectively,they were higer than those in healthy controls and benign breast cancer patients (0.580 and 0. 664 ng•L^-1,P<0.05). The sFas levels in patients with primary cancer before surgery and two weeks after surgery were 0.815 and 0.620 ng•L^-1,respectively, the difference was significant (P<0.05). Ths sFas levels in primary cancer patients increased with tumor stages, the difference was significant (P<0.05), but had no correlation with estrogen receptor (ER), progestogen receptor (PR), and local lymphatic metastasis (P>0.05). Conclusion The serum sFas level in patents with breast cancer may be used as a index to judge the development and prognosis of breast cancer.

Objective To investigate the expressions of CA125 and CA19-9 in endometriosis and the relationship with the serum values of CA125 and CA19-9. Methods Serum CA125 and CA19-9 levels were measured by using the magnetic separated enzyme linked immunosorbent assay respectively in 20 patients with sendometriosis. Forty paraffin samples of endometriosis（20 ectopic endometria and 20 endometria）were examined by immunohistochemical staining method to detect the expressions of CA125 and CA19-9. Results CA125 and CA19-9 were mainly expressed in the cytoplasm of glandular epithelial cells. The positive expression rates of CA125 in ectopic endometrium and endometrium were 90.0% and 25.0%, respectively；The expression rates of CA19-9 in ectopic endometrium and endometrium were 85.0% and 10.0%, respectively. The mean serum values of CA19-9 and CA125（U•mL^-1）in 6 patients with stage Ⅰ and Ⅱ endometriosis were (29.64±10.24) and (25.85±9.45),respectively; the positive rates were 16.7% and 33.3%,respectively. The mean serum value of CA19-9 and CA125 （U•mL^-1） in 14 patients with stage Ⅲ and Ⅳ endometriosis were (46.54±14.29 ) and (58.02±20.11), respectively；the positive rates were 42.8% and 71.4%, respectively. The expression of CA19-9 in ectopic endometrium was correlated to the serum value of CA19-9 （r_s=0.36,P<0.05）, but not CA125 (r_s=0.12,P＞0.05). Conclusion The CA125 and CA19-9 proteins express highly in ectopic endometrium, and express lowerly in endometrium. The main resource of serum CA19-9 in patients with endometriosis is ectopic endometrium.

Objective To investigate the changes of arterial structure and function in patients with hypertension. Methods Structures and functions of aortic, carotid, brachial, radial, ophthalmic and retinal arteries were measured by Doppler ultrasound technique in 55 patients with mild to moderate hypertension. 24 normotensive subjects were served as control. Results The dimension (Ds), intima-media thickness (IMT) and IMT/D radial, resistance index (RI) and stiffness (S) were increased, while the peak velocity in systole (PVS) and maximal shear rate (SR), cross-sectional compliance (CSC) and distensibility in aortic, carotid and brachial arteries were decreased in hypertensive patients compared with controls (P<0.05 or P<0.01). IMT,IMT/D radial,and RI of radial artery were higher in hypertensive patients than those in control group (P<0.01 or P<0.05). The blood flow velocity of the end-diastolic phase of ophthalmic and central retinal artery were distinctly decreased，while RI was increased in hypertensive patients compared with controls(P<0.05 and P<0.01).There were no changes of PI in aortic, carotid, brachial, radial, ophthalmic and central retina arteries (P>0.05). Conclusion There are changes in arterial structure and function in mild-to-moderate essential hypertension, which are more significant in the vascular remodeling of large and moderate arteries.

Objective To determine the progesterone receptor (PR), estrogen receptor (ER), and Bcl-2 protein expressions in endometrial carcinoma and their clinical significances. Methods The expressions of PR, ER, and Bcl-2 in a total of 104 frozen tissues of endometrial carcinoma and 30 cases of paracarcinoma tissues were examined by immunohistochemistry. Results ①The expressions rates of PR, ER, and Bcal-2 protein in endometrial carcinoma were 65.38%, 46.15%, and 50.84%, respectively. They were 66.66%, 60.00%,and 26.66%, respectively, in parcarcinoma tissues. Statistically difference was found in the expressions of Bcl-2 between two groups (P<0.05), while it was not found in the expressions of ER and PR. ②Expression of PR was positively correlated with that of Bcl-2 (r=0.216，P<0.05). There was no relation between ER and Bcl-2. ③Positive expression rate of ER in 16 lymph node metastasis cases was 50.00%, those of PR and Bcl-2 were 12.50% and 25.00%,respectively. Positive expression rates of ER, PR, and Bcl-2 in 88 non lymph node metastasis cases were 45.45%, 75.00%,and 59.09%,respectively. Expressions of PR and Bcl-2 had statistically differences in the cases of lymph node metastasis or non-metastasis　(P<0.05), while ER had not.　④Expression of PR was higher in highly differentiated endometrial adenocarcinoma than that in poorly differentiated one. Conclusion Positive expression rate of Bcl-2 in endometrial carcinoma is high, it is positively correlated with PR expression. PR and Bcl-2 positive expression rates in lymph node metastases cases are low. PR and Bcl-2 might be available prognostic factors.

Objective To evaluate the effects of Panax Notoginseng Saponins on P-selectin (Ps), tissue-type plasmiogen activitor (t-PA) and plasminoge activitor ihibitor-1 (PAI-1) levels in patients with acute coronary syndrome after intracoronary stent implantation. Methods Forty four patients with successful intracoronary stent implantation were divided into routine treatment group (control group,n=22)，routine combined with Lu-Lu-Tong (including Panax Notoginseng Saponins 25 g•L^-1) treatment group (treatment gorup,n=22) at random. For each case, 6 mL elbow venous blood were collected 24 h before implantation, 1 d and 1 month after implantation, and then the serum Ps, t-PA, and PAI-1 levels were detected by ABC-ELISA. Results The levels of Ps （9.170±1.500）g•L^-1 and PAI-1（0.584±0.075）kAU•L^-1 1 month after implantation were both significantly decreased (P<0.05), compared with 1 d after implantation. While the level of plasma t-PA （0.877±0.132）was significantly increased compared with control group. Conclusion Panax Notoginseng Saponins are beneficial to the patients after intracoronary stent implantation through regulating the t-PA/PAI system.

Objective To study the expression of matrix metalloproteinases-9 (MMP-9) in transitional cell carcinoma of bladder（TCCB） and its significance. Methods Immunohistochemistry and in situ hybridization were used to detect the expressions of MMP-9 in 30 surgical resected specimens from patients with TCCB (experimental group) and 5 normal bladder tissues(control group). Results MMP-9 had different degree expressions in experimental group, but the expression was weak or negative in control group. Furthermore, in different grades and stages of tumor tissue, the positive expressions of MMP-9 were significantly increased with stage and grade, there were significant differences in various groups (P<0.05,respectively). Conclusion The expression of MMP-9 is significantly correlated with the pathological grade and stage of TCCB, it could be used as an important index to judge the aggressive and metastatic biologic behavior of TCCB in clinic.

Objective To observe the protective effects of Shengmai inject on on ischemic myocardium in patients with acute myocardial infarction (AMI). Methods Forty-eight AMI patients were randomly divided into Shengmai injection treatment group (n=26) and non-Shengmai injection treatment group (n=22), and 24 normal healthy subjects were used as controls. The levels of MDA and SOD in serum were assayed with colorimetric methods. Results The levels of SOD in all AMI patients were obviously lower than that in normal healthy sujects before therapy, and the levels of MDA were significantly increased (P<0.01); as the time went on, the level of SOD was increased gradually in Shengmai injection treatment group, and MDA level was decreased gradually. Although the levels of MDA and SOD in non-Shengmai injection treatment group had the same trends with the Shengmai injection treatment group, but there was marked difference of the serum content between the two groups (P<0.01). Conclusion With the effects of anti-oxygen-derived free radicals and anti-lipid peroxidation, Shengmai injection has protective effects on the ischemic myocardium in patients with AMI.