To investigate the effects of pEgr-hPTEN stable transfer combined with irradiation on the cell cycle progression and the expression of cell cycle kinase inhibitor P27^kip1 protein of SHG-44 human glioma cells. Methods pEgr-hPTEN vector containing the exogenous wild type PTEN gene was transfected into SHG-44 cells under mediation of lipofectamine in vitro, the positive cell clones were selected and amplified by using G418.Western blotting was used to measure the expression of PTEN protein. Transmission electron microscope was adopted to detect the cell ultrastructural changes and flow cytometry was adopted to analysis the changes of cell cycle progression and the expression of P27^kip1 in SHG-44- sPTEN cells followed by different doses of X-ray irradiation. Results Egr-1 promoter could be induced and activated by irradiation and then enhanced the expression of downstream PTEN gene within 5Gy. The ultrastructure of SHG-44- sPTEN cells had many degenerative changes and many early apoptotic changes including the chromosome condensate around the nuclear envelope. pEgr-hPTEN stable transfer combined with X-ray irradiation could significantly induce G₁ arrest. The expression of P27^kip1 proteins increased in SHG-44-sPTEN stable transfected cells. Conclusion PTEN stable transfer combined with irradiation can significantly induce G₁ arrest. The molecular basis may be correlated with the enhanced expression of PTEN induced by irradiation and increased expression of cell cycle kinase inhibitor P27^kip1.

To construct the mutants of the four serine phosphorylation sites(104,106,118 and 167) in ERα AF-1 and detect their effects on the transcriptional activity of Erα in 293T cells. Methods The coding sequences of the ERα mutants were amplified by recombinant PCR with pcDNA3-ERα as a template and fused in frame with the coding region of FLAG in the pcDNA3-FLAG vector. The fusion proteins were characterized by Western blotting.The effects of the mutants on the transcriptional activity of ERα were determined. Results ERα mutants were successfully constructed and expressed in 293T cells. The transcriptional activities of ERα and its mutants were 3.40,3.21,3.02,3.00,3.54 times of pcDNA3 in the absence of E2. Only the mutants of 118 and 167 phosphorylation sites made the transcriptional activity reduce to 50% of ERα in the presence of E2. Conclusion Only 118 and 167 phosphorylation sites are necessary during the regulation of estrogen on target gene transcription among the four serine phosphorylation sites(104,106,118 and 167) in ERα AF1.

To investigate the mitogenic effects of urotensinⅡ(UⅡ) on renal tubular epithelial cells in rats. Methods The renal tubular epithelialcells in rats were collected by mechanical and enzyme digestive methods and were cultivated and characterized; using immunocytochemistry to investigate bromodeoxyuridine (BrdU) incorporation of DNA in renal tubular epithelial cells of rats; the effects of UⅡ with different concentrations（0,10^-10,10^-9,10^-8,10^-7and 10^-6 mol•L^-1）on proliferation in rat renal tubular epithelial cells were observed using methyl thiazolyl tetrazolium assay (MTT assay) and BrdU incorporation was applied to idetect DNA synthesis in the cells. Results Cytokeratin-18 immunocytochemical staining showed that yellow granules with different intensities and uneven distribution were localized in nucleus surrounding and cytoplasm of the most renal tubular epithelial cells. Yellow granules were deposited in nuclei of cultural renal tubular epithelial cells by immunocytochemical staining following BrdU incorporation. The A values of MTT assay and BrdU incorporation in 10^-10mol•L^-1 UⅡ group were not significantly different from those in control group. In 10^-9and 10^-8 mol•L^-1 UⅡ groups, the A values of MTT assay and BrdU incorporation were significantly higher than those in control group (P<0.01 or P<0.05). Compared with control group, the A values of MTT assay in 10^-7and10^-6 mol•L^-1 UⅡgroups were significantly higher (P<0.05), which was weaker than that difference in 10^-9mol•L^-1 and 10^-8 mol•L^-1 groups, BrdU incorporation were not significantly different. Conclusion UrotensinⅡ reveals a mitogenic effect on rat renal tubular epithelial cells in vitro.

To observe the effect of advanced glycosylation end products (AGEs) on gene expression of connective tissue growth factor (CTGF) in NIH Swiss mouse embryo fibroblasts(NIH/3T3),and to assess the intervention actions of aminoguanidine(AG) and puerarin(Pue) on CTGF mRNA expression in NIH/3T3.Methods AGEs were synthesized by coincubation of BSA with glucose.The AGEs content was measured by fluorescence spectroscopy.NIH/3T3 cells were treated with AGEs (prepared with 20，50，80 mmol•L^-1 glucose) for 24 h.The NIH/3T3 cells were treated with AGEs (prepared with 50 mmol•L^-1 glucose) for 0，6，12，24 and 48 h.The intervention actions of AG and Pue with different concentration (0.25，0.5，1.0 and 1.5 g•L^-1) were evaluated.The CTGF mRNA expression in NIH/3T3 was determined by RT-PCR. Results Compared with BSA control,the CTGF mRNA expression levels in NIH/3T3 were increased by treatment with AGEs (prepared with 20, 50, 80 mmol•L^-1 glucose) for 24 h (P<0.01),among them 50 mmol•L^-1 glucose was highest.Compared with 0 h,the CTGF mRNA expression levels in NIH/3T3 were increased by treatment with AGEs (prepared with 50 mmol•L^-1 glucose) for 6,12,24,48 h (P<0.01),among them 24 h was highest.Compared with AGEs (prepared with 50 mmol•L^-1 glucose) for 24 h,AG and Pue with different concentrations significantly decreased the CTGF mRNA expression in NIH/3T3 (P<0.01).Conclusion AGEs up-regulates CTGF mRNA expression in NIH/3T3,which is related with glucose concentration and acting time.Based on the above mentioned,AGEs may promote the fibrotic process of diabetic complications.AG and Pue can attenuate the profibrotic effects of AGEs.

To investigate the protective effects of Astragaloside Ⅳ(ASⅣ )on cardiomyocytes against hydrogen peroxide(H₂O₂)-induced injury. Methods Cultured neonatal rat cardiomyocytes were divided into control group;0.1%DMSO solven group；H₂O₂ group, in which cells were treated with H₂O₂ ( 0.2　 mmol•L^-1) for 24 h;ASⅣ+H₂O₂ groups, in which cells were pretreated with ASⅣ (with final concentration of 3,10,30 mg•L^-1) for 30 min before H₂O₂ treatment; Astragaloside injection group (150　g•L^-1 ).Morphological changes of myocardial cells damaged for 24 h by 0.2 mmol•L^-1 H₂O₂ under the inverted microscope were observed. The cardiomyocyte viability was analysed by MTT assay; lactate dehydrogenase (LDH) activity, superoxide dismutase ( SOD) activity, malonaldehyde (MDA) content and nitric oxide (NO) content were detected in culture media. Results Most of cells in H₂O₂ group were crenulated,the nucleus were dim and the pseudopodia were disappeared. The degrees of lesion in ASⅣ (3,10, 30　mg•L^-1) groups were lessened obviously, the nucleus were bright and the the pseudopodia were thinned slightly. Cardiomyocyte viabilities in ASⅣ (3,10, 30　mg•L^-1) groups were higher than that in H₂O₂ group (P<0.05 or P<0.01).LDH activity and MDA content in ASⅣ (10, 30 mg•L^-1)groups were lower than those in H₂O₂ group (P<0.05 or P<0.01), and SOD activity and NO content in ASⅣ (10, 30 mg•L^-1)groups were higher than those in H₂O₂ group (P<0.05 or P<0.01). Conclusion ASⅣ can protect cardiomyocytes from H₂O₂ injury by improving cell antioxidant ability and increasing NO content.

Abstract:Objective To study the inhibitory effect of methylmercury chloride (MMC) on rat C6 glioma cells in vitro. Methods The rat C6 glioma cells were cultivated in vitro and divided into control group and MMC-treated group (0.08－10.00 μmol•L^-1 MMC were divided into 8 groups with concentration gradient). MTT assay was performed to evaluate the proliferation inhibitory effect and cytotoxicity effect of MMC with different concentrations on cultured rat C6 glioma cells, and flow cytometry was used to assess the effects of MMC treatment on cell apoptosis and cell cycle in rat C6 glioma cells. Results 1.25, 2.50, 5.00 and 10.00 μmol•L^-1 MMC could inhibit the proliferation of cultured rat C6 glioma cells in vitro, the viabilities of MMC treated C6 glioma cells were significantly lower than those in control group (P<0.05), the inhibitory effect was in a dose-dependent manner. The cell viabilities of C6 glioma cells treated with 2.50, 5.00 and 10.00 μmol•L^-1MMC for 4, 8, 16 and 32 h were significantly lower than those in control group (P<0.05), the cytotoxicity effect was in a dose and time dependent manner. When C6 glioma cells were treated with 0.31, 0.63, 2.50, 5.00 and 10.00 μmol•L^-1 MMC for 24 h, the apoptosis/necrosis rates were significantly higher than those in control group (P<0.05); when treated with 1.25, 2.50 and 5.00 μmol•L^-1 MMC for 72 h, the rates of G₀/G₁ phase cell significantly increased compared with control group(P<0.05); while cells into S phase were decreased(P<0.05). Conclusion MMC has cytotoxicity effect on cultured rat C6 glioma cells in vitro, and can inhibit proliferation and induce apoptosis.

To evaluate the activity of novel polyoxometalate against hepatitis B virus (HBV) in vitro. Methods MTT assay was used to evaluate the growth inhibitory effects of polyoxometalate on 2.2.15 cell lines, Hepatitis B e(s) antigen (HBeAg) and Hepatitis B surface antigen in the culture medium were determined by ELISA. Southern blotting was performed to detect HBV DNA in Hep G2 2.2.15 cells. Real-time quantity PCR was applied to detect the contents of introcellular HBV mRNA and extracellular HBV DNA. Results The median toxicity concentration (TC₅₀) was 1590.46 mg•L^-1 on 2.2.15 cells. The inhibitory rats of PTW-6 on HBeAg and HBsAg were markedly higher compa red with control group (P<0.05). The median inhibitory concentration (IC₅₀) of PTE-6 in extracellular HBV DNA and introcellular HBV mRNA were 51.1 and 63.6 mg•L^-1, respectively. Conclusion Novel polyoxometalate is a potential compound for treatment chronic HBV infection.

To investigate the anti-tumor activity of ADM-fiber on mo use transplanted hepatoma. Methods The ADM-fiber was implanted into H22-bearing C57BL mice. They were divided into four groups: experimental group (ADM-fiber group ), control 1 group (ADM intratumorally injection), control 2 group(ADM intravenous injection), control 3 group(physiologic saline intravenous injection). The tumor volume was calculated on the 1st,3rd,6th,9th,13th day after administration. The tumors were dissected on the 14th day. This biopsy was weighted and the tumor inhibitory rate was then calculated as formula. Biopsy was then observed with HE staining, analyzed with immunohistochemistry method on the expression of apoptosis relative proteins caspase-3/fas, and the apoptotic rate was analyzed using flow cytometer. Results The growth of tumor volume was slower in experimental group and control 1 group on the 6th day than those in other groups (P<0.05), it was slower in experimental group than those in the control 1 group on the 9th,13th day, and it was obviously slower than those in the control 2 and 3 groups on the 9th and 13th day;the inhibitory rate of tumor growth of experinental group was significantly higher than those in the others（P<0.01）; the necrosis of tumor in experimental group was serious than other groups, and the expression of caspase-3/fas was increased numerously in the experimental group(P<0.01); compared with control groups,the apoptotic rate was also enhanced substantially in experimental group(P<0.01）. Conclusion ADM-fiber embedded can exhibit significantly tumor inhibition on mouse H22 transplanted hepatoma, which illustrates that embedding retarders can significantly accelerate the apoptotic rate of tumor cells through ADM-fiber system.

To study the expressions of IGF-Ⅰ and TGF-β₁　in lung tissue of rats with pulmonary fibrosis. Methods Sixty male Wistar rats were randomly divid ed into two groups: control group (n=20) and experimental group (n=40), and poured respectively by saline and bleomycin solutions, and killed on the 7th, 14th, 21th, 28th day. Immunohistochemistry method was used to analyze the expressions of IGF-Ⅰ and TGF-β₁ in lung tissue. Results After 4 weeks induction by bleomycin solutions, the lung tissue was obtained and stained by HE, the pathological changes were in accordance with pulmonary fibrosis. Pulmonary interstitial fibers were found by VG staining. So lung fibrosis models induced by bleomycin were set up successfully. The expressions of IGF-Ⅰ and TGF-β₁ were increased during the progression of pulmonary fibrosis. Excellent differences of percentage of positive cells and expression level of IGF-Ⅰ and TGF-β₁ were found between control group and experimental group（P<0.05).The expressions of IGF-1 were increased on the first, second, and third week, and decreased on the fourth week, however, there were excellent differences between the first and third week and between the first and fourth week（P<0.05）. The peak of TGF-β₁ expression was on the second week, and decreased during the third week. But its expression on the fourth week was also higher than that on the first week. Excellent differences were found between the first week and the second or third week（P<0.05）. Conclusion The high level expressions of IGF-Ⅰand TGF-β₁ have the vital significance for the formation of pulmonary fibrosis, both IGF-Ⅰ and TGF-β₁ also take an effect on the pulmonary fibrosis formation, and may have synergetic effect.

To observe the effects of porous polylactic acid/polyglycolic acid copolymer (PLGA) filling into extraction socket on the regeneration of alveolar bone in rats. Methods Sixty male Wistar rats were divided into experimental group and control group (n=30). PLGA scaffold was immediately implanted in the mandibular incisor root sockets after removal of incisor teeth. Soft X-ray photography, structural observation, light microscopy were used to evaluate the effects of composite on bone healing in a rat tooth extraction socket. Results The relative lengths of residual alveolar ridge in the experimental group 4,8 weeks after operation were shorter than that in the control group，and there were significant differences between the experimental group and the control group (P<0.05)．The relative width of residual alveolar ridge in the experimental group 2,4,8 weeks after operation were higher than that in the control group，and there were significant differences between the experimental group and the control group (P<0.05)．Histologic examination on the 8th week after operation showed that there was little undegradable scaffold in the experimental group, while the tooth socket in the control group was filled with new borne bone, and there was space between the new bone; the materials in the experimental group was degraded completely on the 12th week after operation, the woven bone was seen both in the experimental group and the control group. Conclusion The implantation of a degradable hydrophobic polymer does not significantly alter the procession of bone healing, and it can preserve the length and width of residual ridge. The new material is of good biocompatibility, biodegradability, stability, and osteoconduction.

To assess the efficacy of the improved lentiviral vector containing gene enhanced green flurosecent protein (EGFP) delivery pathway in nervous system disease. Methods The cDNA encoding EGFP was cloned by PCR with pIRES2-EGFP as the template. The resulting gene of EGFP was subcloned into the site BamH Ⅰand XhoⅠof pLenti6/V5 TOPO by using DNA recombinant technique. It was introduced into 293ET cells by Ca3(PO4)2 methods using four plasmids. The improved four-plasmid system was made up of the vector plasmids which consisted of EGFP, the packaging plasmid pLp1 and pLp2, the envelope plasmid encoded the vesicular stomatitis virus-G glycoprotein (VSV-G). 72 h after transfection, the viral supernatant on 293ET cells was collected. NIH 3T3 cells were infected with the rLent/EGFP and the fluorescence was detected. The titers of the lentiviral vector were determined by positive rate of EGFP cells by means of FACS. rLent/EGFP was transfected into the primary neocorticall cells of SD rats, the expression of EGFP was observed by confocal microscopy. Results The EGFP cDNA cloning and pLent/EGFP construction were confirmed by the evidences of DNA sequence analysis and restriction enzymes digestion. The transfected NIH 3T3 cells were found containing strong expression of EGFP, confirming that the four-plasmid system of the lentiviral vector and its packaging cell line as well, were successfully constructed. The titer of the rLent/EGFP was 2×10⁶. 72 h after transfection, the higher fluorescent intensity of EGFP was found in the primary neocorticall cells under confocal microscopy. Conclusion The recombinant Lent/EGFP is successfully constructed by the improved four-plasmid system. It could infect the non-dividing mammalian cells, as the primary neocorticall cells.

To systematically compare the effects of integrated Chinese-Western medicine(ICWM) with Western medicine alone(WM) in treatment for type 2 diabetes mellitus(T2DM). Methods Both a computer-aided search of PubMed,CNKI, VIP, Wanfang data and an intensive search by hand were conducted to identify all randomized controlled trials assessing the effect of ICWM with WM in treating T2DM and Meta-analysis was performed. Results Ninety six studies　were collected,of which 20 studies were involved.Combined OR of efficient rate was 4.26 and its 95% confidence interval (CI) was 3.42-5.32,weighted mean difference (WMD) of fasting blood glucose(FBG), triglyceride and total cholesterol were －1.16 mmol•L^-1［95%CI －1.36，－0.96］, －0.27 mmol•L^-1 ［95%CI －0.41，－0.12］, －0.71 mmol•L^-1 ［95%CI －0.98，－0.44］,respectively. Conclusion ICWM has a better effect in treating T2DM compared with WM alone.

To isolate, cultivate and identify the human embryonic fibroblasts (hEFs) derived from the gonadal ridges and dorsal mesenteries of human embryos, and to detect the expression by hEFs of cytokines crucial for the growth of human embryonic germ cells (hEG) in vitro. Methods The hEFs were isolated by enzyme digestion from gonadal ridges and dorsal mesenteries of 5- to 9- week old human embryos. The cells were then cultivated. The biologic characteristics (morphology, growth characteristics and cell cycle) of these cells were also studied. The reverse transcription-polymerase chain reaction (RT-PCR) was used to seek the expressions of a specific fibroblast marker, prolyl 4-hydroxylase β, and a specific marker of epithelial cells, cytokeratin-4, in the cells. The expressions of cytokines essential for the growth of hEG, namely basic fibroblast growth factor (bFGF) and leukemia inhibitory factor (LIF), were also examined by RT-PCR. Results The hEFs were successfully isolated and cultivated from gonadal ridges and dorsal mesenteries of human embryos. They could be passaged beyond the 25th generation. The biologic characteristics of the cells did not change, even in high-passage cells or frozen-thawed cells. The cells expressed prolyl 4-hydroxylase β, but not cytokeratin-4, which was similar to the fibroblasts. The cultured cells expressed bFGF and LIF. Conclusion The hEFs derived from gonadal ridges and dorsal mesenteries of human embryos are successfully isolated and cultivated,and the cells express the cytokines essential for the growth of hEG in vitro.

To investigate the pathogenetic mechanism of ubiquitin-proteasome dysfunction in a model of Parkinson’[KG-3]s disease (PD), which can provide the theoretical basis for PD. Methods After establishment of PD model induced by PSI in PC12 cells, proteins of untreated (DMSO) and PSI-treated PC12 cells were extracted 36 h after incubation, and then the maps of the extracted proteins were established by DIGE system. The altered protein spots were identified with MALDI-TOF Pro MS and database searching. Results Thirty-six treatment of PC12 cells with PSI induced the appearance of cytoplasmic Lewy body-like eosinophilic inclusions and apoptosis. The percentage of apoptotic cells was 25.53%. ERp29 were identified by MALDI-TOF Pro MS. The expression of ERp29 decreased in treatment group, compared with normal group（P<0.01）. Conclusion Endoplasmic reticulum stress (ERS) may play an important role in the pathogenesis of ubiquitin-proteasome system dysfunction induced PD.

To study the effect of methylmercury chloride(MMC) on proliferation and apoptosis in leukemia cell lines NB4 and K562. Methods NB4 and K562 were treated with 1.25,2.50,5.00,10.00 and 20.00 μmol•L^-1 MMC and asenic troxide (ATO) for 0,6,12,24,48 and 72 h,the cell viability rate was detected by MTT assay; the apoptosis was observed with fluorescence microscope after stained by Hoechst 33258. Results The viability rates of NB4 and K562 cells treated with 10 μmol•L^-1 MMC for 24 h were 22.0% and 70.0%；when treated with 10　μmol•L^-1 ATO for 24 h the viability rates were 24.4 % and 52.0%; compared with control group,there was significant difference (P<0.05). The significant apoptostic picture was obtained with fluoresce nce microscope. The two drugs had the similar effect when they were used to treat NB4 and K562 cells in the same dose and for the same time. Conclusion MMC can induce apopt osis of NB4 and K562 cells and inhibit proliferation of leukemia cell lines NB4 and K562 significantly and the effect is in time- and dose-dependent manner.

To study the effect of simulated cardiac microenvironment in vitro on the differentiation of human marrow mesenchymal stem cells (hMSCs) to cardiomyocytes (CM). Methods hMSCs were isolated and expanded in culture and the phenotypes (CD34, CD44 or CD45) of hMSCs were identified by fluorescent - activated cell sorting ( FACS ). The parallel control groups were without mono-antibody. Fifth passage hMSCs, having good growth characteristics and the purity> 95%, were co-cultivated with CM, which derived from 2-day neonatal SD rats as simulating of the cardiac microenvironment. Expression of cardiac-specific marker cTnI, identified by immunohistochemistry, was used as a marker of cardiac muscle differentiation. Results hMSCs did not express CD34 and CD45; but it expressed a high level of CD44,the positive rate of CD44 in hMSCs group （93.26%±2.48%) was higher than that in parallel control group （3.42%±1.09%）(P<0.01). After co-culture of hMSCs and CM, the morphology of hMSCs changed closely into that of CM, and the cells demonstrated positive staining for cTnI. Conclusion hMSCs have the potential to differentiate into cardiomyocytes under the simulated cardiac microenvironment in vitro.

To construct a CTL epitope vaccine against human papillomavirus and to express it in E. coli. Methods The gene sequences encoding CTL epitopes selected by computer-assisted epitope prediction were synthesized by PCR and sub-cloned into pET28a expression vector. The recombinant plasmid pET28a-Tat-HPVepi was transformed to E. coli for expression and the expressed protein was identified by Western blotting. Results The DNA sequences of selected CTL epitopes were obtained by 5 rounds of PCR, the constructed recombinant plasmid pET28a-Tat-HPVepi was effectively expressed in E. coli. The expressed products，identified by Western blotting，showed specific bands. Conclusion The CTL epitope vaccine against human papillomavirus is successfully constructed and express in E. coli.

To establish anti-CD₃ monoclonal antibody activated killer cells (CD₃AK) culture system from 20 mL peripheral blood and observe the cytotoxity against tumor cells in vitro . Methods Anti-CD₃ monoclonal antibody and recombinant rIL-2 costimulated human peripheral blood mononuclear cells (PBMC) for inducing CD₃AK. CD₃AK was obtained and cultivated in vitro for a long time. The biological characteristics, proliferative kinetics, cytotoxicity against tumor cells and the phenotype changes of CD₃AK were analyzed. Results In the cultural period of 7-22 d,the proliferative multiple of CD₃AK increased significantly and outstandingly up to 317 times.Immunophenotypic analysis showed CD₃⁺ cells increased from 51.9% before the activation to 94.6%.CD₄⁺ cells increased from 19.5% to 51.1%. CD₈⁺ cells increased from 22.1% to 52.1%.In addition, the cytotoxicity activity of CD₃AK was more significant with E/T ratio of 20∶1 group and 40∶1 group than 10∶1 group.The best antineoplasmic activity time was in the period of 7-9 d. Conclusion CD₃AK,which is induced from peripheral blood,is a heterogeneity group of CD₃⁺,CD₄⁺ and CD₈⁺ T cells and has significant antineoplasmic activity in vitro.

To study the apoptosis of H9c2 myocardial cell lines induced by different concentrations of cadmium chloride(CdCl₂).Methods The H9c2 cells were exposed to 5,10,30,50 and 80 μmol•L^-1 CdCl₂ for 6,12 and 24 h,the survival rate was measured by MTT assay and the apoptotic rate was detected by Annexin Ⅴ-FITC/propidium iodide (PI)-flow cytometry (FCM ) and acridine oringe/ethidium bromide(AO/EB) staining.Results The survival rates in the groups treated with CdCl₂ decreased significantly compared with those in negative controls (P<0.05 or P<0.01). The apoptotic rates in the groups treated with CdCl₂ in creased significantly compared with those of negative controls(P<0.001),but there were no significant differences between various doses groups(P>0.05).The typical morphological changes of early apoptosis in H9c2 cells could be observed under fluorescent microscope by AO/EB staining. Conclusion The H9c2 cells are sensitive to toxicity of CdCl₂,low dose and short time treatment with CdCl₂ can induce apoptosis of H9c2 c

To investigate the effects of Taurolidine on the apoptosis of melanoma B16-4A5 cells in mice and its mechanism. Methods The cell growth, apoptosis, Bcl-2 family protein expressions and caspase-9 activity were assessed with MTT method, flow cytometry, Western blotting and ApoTarget kit, respectively. Results Taurolidine had significantly inhibitory effect on B16-4A5 cell growth, and induced the apoptosis in a dose- and time- dependent manner. Taurolidien increased the expression levels of Bax and Bad and decreased the expression level of Bcl-2, and further activated caspase-9. Conclusion Taurolidine can inhibit B16-4A5 cell growth and induce apoptosis by mediation of Bcl-2 family proteins and activation of caspase-9.

To establish a practical model for Wistar rat cochlea organ cultivated in vitro and observe the effect of hypoxia on the inner hair cells (IHC) and outer hair cells (O HC). Methods The cochlear basal membrane from 3-day-old Wistar rats was used.The cochlear basal membrane cells were divided into 8 groups.Among them 7 groups were used as experimental groups.The cells in experimental groups were put in incubator (37℃, 90％N₂, 5％CO₂, 5％O₂)to cultivate with hypoxia for different time. The other group was used as control,the cells were cultivated in incubator( 37℃, 5％CO₂).The cultures were phalloidin-labeled and the number of IHC / OHC were counted in unit area （24 mm×36 mm） and the morphological changes of IHC and OHC were observed under microscope. Results No significant change of IHC was found under hypoxia for 0.5 h. IHC under hypoxia for 1 h appeared light swelling and cell density decreased,but the cell density in unit area had no remarkable difference compared with control (P>0.05). IHC loss was found under hypoxia for 2 h to 48 h in a time dependent manner, the cell density in unit area had remarkable difference compared with control (P<0.01). The phenomena of empty nerve cup was found under hypoxia for 6 h. The OHC loss a nd array disorders appeared under hypoxia for 6 h and severe damage under hypoxia for 48 h, the cell density in unit area had remarkable difference compared with control (p<0.01). Conclusion Hypoxia could cause the hair cell loss in vitro. Moreover IHC is more susceptible to hypoxia than OHC.

To study the role of Schwann cells apoptosis in the pathogenesis of experimental autoimmune neuritis　(EAN) in TNF receptor Ⅰ knock out　(TNFR Ⅰ－/－)mice. Methods For induction of EAN, TNFRⅠ－/－and wild type C57BL/6 mice were immunized by subcutaneous injection into the back with the peripheral nerve P0 protein peptide 180-199; clinical scores of EAN were assessed and scored immediately before immunization (day 0) and thereafter every other day until day 46 post immunization (p.i.). On day 22 p.i., Schwann cells were collected from　the two groups and cultivated in vitro. The expressions of Fas and Annexin-Ⅴ（Annexin-Ⅴ+/PI－）on Schwann cells from TNFRⅠ－/－ and wild type EAN were detected by flow cytometry. Results More light clinical signs of EAN were observed in the TNFRⅠ－/－ mice（1.50±0.19） than in wild type mice（1.90±0.16）; the percentage of Fas expression on Schwann cells was significantly decreased in TNFRⅠ－/－ mice（88.03%±1.40%)as compared with wild type mice（94.70%±1.53%)(P<0.05). An decreased tendency of Annexin-Ⅴ（Annexin-Ⅴ+/PI－）expression on Schwann cells was seen in TNFRⅠ－/－ mice（8.78%±0.94%)as compared with wild type mice（13.03%±4.15%), but no significant difference. Conclusion TNFRⅠ may induce peripheral nerve injury and aggravate EAN through increasing Schwann cells apoptosis.

To investigate the regulatory effect of curcumin on proliferation inhibition and apoptosis in human prostate carcinoma PC-3M cells.Methods The PC-3M cells were divided into 4 treatment groups (10,20,30 and 40 μmol•L^-1 curcumin) and control group. The morphological alterations of PC-3M cells after treated for different time were observed by using inverse microscopy,the inhibition of cell proliferation was detected by MTT assay, the cell cycle phase was analyzed by flow cytometry,the apoptosis was inspected by acridine orange-ethidium bromide fluorescent staining method,the protein level of caspase-3 in carcinoma cells was observed by SP immunohistochemistry. Results After being treated with curcumin, PC-3M cells grew round and small obviously and were against the wall under the inverse microscope. with the increasing of the concentration of cucurmin and the elongation of the treated time,the PC-3M cell growth was inhibited,the inhibition of proliferation of PC-3M cells increased in a time-and dose- dependent manner（P<0.01）;partial cells presented the morphological changes of apoptosis under the fluorescent microscope,the apoptotic rates of the control group and 4 treatment groups were（9.83±1.53）%,（19.50±1.00）%,（24.83±2.52）%,（30.17±2.08）% and（38.50±2.65）%, respectively;the cell number in S and G₂/M phase were increased, the cell number in G₀/G₁ phase was decreased;with the increasing of concentration of curcumin,the caspase-3 expression was increased in a dose- dependent manner（P<0.01）. Conclusion Curcumin could significantly inhibit the growth of PC-3M cells, the induction of apoptosis through up-regulating caspase-3 is probably one of its molecular mechanisms.

To discuss the inhibitory effects of Juglans mandshurica maxim extract(JMME) on S₁₈₀ mouse sarcoma and its immunity accommodation.Methods The mice bearing sarcoma in three groups(n=10)were aderminastered with JMME with doses of 0.023,0.045 and 0.090　g•kg^-1•d^-1by gavage. Positive and negative control groups were set up.The inhibitory rate of JMME on S₁₈₀ mouse sarcoma was detected. The cell culture was used to observe the inhibitory effect of JMME on the sarcoma, the influences of JMME on the tumor necrosis factor(TNF) activity and the interleukin-2(IL-2) luring level were observed with MTT. Results JMME had inhibitory effect on the sarcoma. The inhibitory rates in JMME groups with different does were 32%,43%,56%,respectively,there were significant differences compared with negative group（P<0.05）. The inhibitory rate of JMME on tumor cell cultivated in vitro (60.10%)was higher than that in control group（P<0.05）. The TNF activity and IL-2 luring level in JMME group were higher than those in control group（P<0.05）. Conclusion JMME has significantly inhibitory effect on S₁₈₀ mouse sarcoma.Its mechanism is associated with inhibition of the growth of the exosomatic tumor and regulation of immunity function of the body.

To investigate the inhibitory effect of sodium phenylbutyrate (SPB) combined with gefitinib on NB4 cells.Methods The NB4 cells were divided into 8 groups randomly:control group,PMA group,H-7 group,SPB+PMA group,gefitinib group,gefitinib+PMA group,SPB+gefitinib group.The inhibitory rate of medicine was detected by MTT，the apoptosis ofNB4 cells was detected by flow cytometry, the expressions of PKC,CDK, and P53 were detected with Western blotting method. Results The NB4 cells were suppressed obviously. The rate of growth inhibition and proliferation index in SPB+gefitinib group were 92.8% and 5.96%, those in SPB group　were 45.1% and 29.6%,and gefitinib group were 61.0% and 35.9%, the differences were significant(P<0.05）.The expression of P53 in SPB+gefitinib group increased but CDK₄ and p-PKC decreased,compared with curement only, the difference was significant(P<0.05).Conclusion SPB combined with gefitinib can significantly increase the apoptotic rate and inhibitory rate of tumor cells than SPB or gefitinib only, its mechanism is suppressing PKC to block the cell cycle.

To investigate the reveral effect of Tanshitone(Tan) Microemulsion on multidrug resistance(MDR) of sensitive K562 cell line(KS) and ADM resistant K562 cell line (KA) and the mechanism. Methods The reversal effect of Tan Microemulsion (Tan and Microemulsion as controls) on MDR of KA cells was observed. The inhibitory effect of cell growth was investigated by MTT. The apoptosis of KS and KA cell lines was observed by flow cytometry.The intracellular drug concentration was measured by detecting fluorescent density. Results The result showed that Tan Microemulsion，Microemulsion and Tan reduced the expression levels of P-gp and Bcl-2 in KA cells and Tan Microemulsion had the most significant effect. The MDR reversal folds of Tan Microemulsion of KA was highest among all 3 groups（P<0.05）. The intracellular concentration of ADM improved significantly by Tan Microemulsion, Microemulsion and Tan. The intracellular concentration of ADM treated by Tan Microemulsion was higest(P<0.05）. Conclusion Tan Microemulsion can reverse the MDR of KA cells to some extent and increase the ADM concentration of KA cells.

To construct PCI-hepatocyte growth factor (HGF) expression vector and to detect its transient expression in transfected COS₇ cell line. Methods Techniques of gene recombination and gene transfection were employed. Human HGF cDNA was ligated to PCI-neo. The recombinant plasmid was transfected into COS₇ cell line with liposome. The expression of HGF protein was observed by ELISA method. Results PCI-neo-HGF expression vector was analyzed by transient expression，the fragment was same as theoretical value. Compared with GenBank, the conspecific property of HGFcDNA array was 99%. Identification of PCI-HGF by enzyme digestion showed that HGF fragment had been cloned into BamHⅠand SalⅠ sites of PCI-neo vector. The expression of HGF was observed in COS₇ cells transfected with PCI-HGF for 12 h; the value of expression was 2 000　ng•L^-1 in 36-72 h, then,the value gradually declined. Conclusion The PCI-HGF expression vector is constructed successfully and it can express in transfected COS₇ cell line.

To detect the partial molecular characteristics of a novel fibrinolytic protease from the coelomic fluid of Nereis virens identified and purified to homogeneity. Methods The fibrinolytic protease was purified with anion and cation exchange chromatography and gel filtration chromatography. The identification of fibrinolytic activity and active distributed curve had been assessed by method of fibrin plate. Its apparent molecular weight and isoelectric point were analysed by two-dimensional gel electrophoresis (2-DE). The effects of several protease inhibitors on the protease activity were examined, and its protease type was identified. Results A novel and single chain fibrinolytic protease was purified effectively. Its apparent molecular weight and isoelectric point were 29 000 and 4.5, respectively. The proteolytic activity peaked at pH 7.8 and 45℃. The protease was completely inhibited by DFP and PMSF, assessin g as a serine protease. Conclusion From the coelomic fluid of Nereis virens, a novel and single chain serine protease with more strong fibrinolytic activity is discovered. The fibrinolytic protease has a medical value for preventing and treating thrombosis.

Abstract:Objective To investigate the genetic association of HLA class II DRB1,DQB1,DQA2 and DQB2 locus with schizophrenia in the Han people of Northern China.Methods The polymerase chain reaction (PCR)and estrictive fragment length polymorphism (RELP) were applied to detect the gene types in the 195 Han family trios including patients with schizophrenia and their healthy parents in the north of China. The chi-square (χ²) test, transmission disequ ilibrium test(TDT) and the haplotype relative risk (HRR) analysis were used to process the genotyping data statistically. Results The genotypic frequency of four gene s did not deviate from Hardy-Weinberg equilibrium in both case and control group s; The SNP rs2239800, a G to A base change, presented in the DQA2 locus. Its allelic frequencies showed significant difference between case and control (χ²= 5.021, P=0.025). And the AA genotype of rs2239800 showed associations with schizophrenia. Conclusion The polymorphism of rs2239800 in the DQA2 gene may be associated with schizophrenia.

Objective To investigate the genetic association of MTHFRC677T between CBS844ins68 polymorphism and young ischemic stroke. Methods The polymorphism of MTHFRC677T and CBS844ins68 in 98 stroke patients and 116 control subjects were examined and analyzed by using PCR-RFLP. Results The frequencies of two alleles were as follows: T allele 54.09%, C allele 45.91% in patients and T 37.93%, C 62.07% in controls （χ²=11.18, P<0.05）. Odds radio of T to C was 1.93（95%CI 1.31-2.84）. Logistic regression analysis showed that incidence of young ischemic stroke in people with MTHFR677C→T was 1.907 times of that in people without the mutation. The significant association wasn’ t found between young ischemic stroke and 844ins68 mutation in CBS gene. MTHFR C677T variation had significant correlation with young ischemic stroke. Conclusion C677T mutation site in MTHFR gene has a significant relation with young ischemic stroke and MTHFR gene is a predisposing gene of young ischemic stroke by Logistic regression analysis. T allele carrier has a higher risk to stroke. 844ins68 mutation site in CBS gene is failed to find any obvious relation with young ischemic stroke.

Objective To investigate the role of the expressions of APC, bcl-2 and c-met gene in the progress of gastric cancer and their significances in the diagnosis of early gastric cancer. Methods The immunohistochemical technique was used to detect the expressions of APC, bcl-2 and c-met gene in 30 cases of human gastric carcinoma(GC),30 cases of intestinal metaplasia (IM),30 dysplasia (Dys) gastric mucosa, 10 gastric adenoma(GA) and 20 normal gastric mucosa. Results ①The positive expression rates of APC in GC,IM, Dys and GA (53.3%,67.7% and 53.3%,respectively) were significantly lower than those in normal gastric mucosa(90.0%,P<0.05).The positive rate of APC in moderate and severe Dys(47.1%) was lower than that in mild Dys(61.5%, P<0.05).The expression rate of APC in GC without lymph node metastasis was higher than those with lymph node metastasis (P<0.05).②The positive expression rates of bcl-2 in GC (66.7%),IM(40.0%) and Dys(43.3%) were higher than those in normal gastric mucosa(5.0%) and GA(10.0%). The positive expression rate of bcl-2 in mild Dys (30.8%) was lower than those in GC(66.7%,P<0.05). The bcl-2 expression showed a significant negative correlation with lymph node metastasis and Lauren type(P<0.05).③The positive expression rates of c-met in GC(76.0%),IM(63.3%) and Dys(60.0%) were higher than those in normal gastric mucosa (10.0%,P<0.05)) and gastric adenoma (10.0%, P<0.05).The positive expression rate of c-met in moderate and severe IM (70.6%)was higher than those in mild IM(46.1%,P<0.05). The positive expression rate of c-met in moderate and severe Dys(75%) was higher than that in mild Dys(55.6%, P<0.05).The expression of c-met gene in GC with well differentiation was higher than that with bad differentiation(P<0.05). The expression of c-met gene in GC with lymph node metastasis was higher than that without lymph node metastasis (P<0.05). Conclusion The lose of APC or bcl-2 and c-met gene overexpression are contributed to the occurance and development of GC.To detect the expressions of APC, bcl-2 and c-met gene in moderate and severe Dys may be helpful for diagnosis of early gastric cancer.

Abstract:Objective To study the distribution and expressions of aquaporin(AQP) 1,2,3 in human cystitis glandularis tissue.Methods The expressions and distribution of AQP1,AQP2,AQP3 in 30 cases of cystitis glandularis tissue and 30 cases of normal bladder tissue were detected by immunohistochemistry technique,and the gray scale values of the dyeing consequence were examined by computer.Results In cystitis glandularis tissue AQP1 mainly expressed in the endothelial cells of micrangium,the immunohistochemical positive exponent was 35.947；AQP2 expressed in the cell membrane and periplasm of the mucosa epithelial cells，the immunohistochemical positive exponent was 34.644；and AQP3 did not express.In normal bladder tissue, AQP1 mainly expressed in the endothelial cells of capillary and arteriolar under the mucous membrane of urinary bladder,AQP2 and AQP3 expressed in the celluar membrane and periplasm of the mucous membrane of urinary bladder.The immunohistochemical positive exponent of AQP1,AQP2,AQP3 in the normal bladder tissue were 57.038，61.425 and 60.468，respectively,there were significant differences compared with AQPs in the cystitis glandularis tissue P<0.01）. Conclusion The expressions of AQPs are obviously reduced in cystitis glandularis tissue,and the inflammation affects the expression of AQPs.

Objective To detect the expression of COX-2 mRNA in astrocytic tumors (ATs) tissue, also to analyze its correlation with the expression of Fas mRNA. MethodsReverse transcription polymerase chain reaction (RT-PCR) technique was used to examine the levels of expression of COX-2 mRNA and Fas mRNA in ATs tissue and non-tumor tissue.Results The rates of of COX-2 mRNA in non-tumor tissue and ATs tissue were 33.3%and 78.4%, respectively. The positive exprssion rates of Fas mRNA in non-tumor tissue and ATs tissue were 8.3% and 74.5%. The expression levels of COX-2 mRNA and Fas mRNA in ATs tissue were significantly higher than those in non-tumor tissue（P<0.05）. There was obvious correlation between expressions of COX-2 mRNA and Fas mRNA（r=0.525,P=0.008）. Conclusion The expression of COX-2 mRNA is correlated with carcinogensis and development of ATs, and it is an effective marker in diagnosis of ATs. A positive correlation between expressions of COX-2 mRNA and Fas mRNA is found, it is speculated that there may be a common transcription mechanism which constructs multiple pathways contributing to the inhibition of apoptosis in ATs.

Objective To explore the effect of steopontin (OPN) as marker in colon cancer tissue and analyze the relationship between osteopontin and recurrence.Methods ELISA method was used to determine the serum levels of osteopontin in 58 patients with colon cancer(observation group) and 22 cases of normal subjects (control group);semi-quantity RT-PCR was used to detect the expression of osteopont in in tissues.The expression level of OPN mRNA in colon cancer tissue and serum OPN level in 23 recurrence cases and 35 non-recurrence cases one year after operation were observed.Results The OPN mRNA expression in colon cancer tissue and the serum osteopontin level in patients in observation group were higher than those in control group(P<0.01). The OPN mRNA expression and the serum level in patients with recurrence one year after operation were higher than those without recurrence(P<0.01).Conclusion Osteopontin is one of the tumor markers and it could be used in diagnosis and prognosis evaluation of colon cancer.

Objective To approach the expressions of vascular endothelial growth factor (VEGF) and transforming growth factor-beta （TGF-β） in periosteum in patients with congenital pseudarthrosis of tibia(CPT) and elucidate the pathogenesis possibility. Methods The expressions of VEGF and TGF-β in 19 specimens from patients with CPT were detected by using immunohistochemical method.Ten normal periosteum from the healing site after tibia fracture were taken as negative group; 15 fresh periosteum from the close tibia fractures were positive group. Results VEGF and TGF-β expressed in vascular endothelial cytoplasm of periosteum. The expression levels of VEGF and TGF-β of CPT were lower than those in positive group（P<0.01）. There were no significant differences of VEGF and TGF-β expressions between normal periosteum and CPT (P>0.05). Conclusion The decreasing of expression levels of VEGF and TGF-β in periosteum may be involved in pathogenesis of CPT.

Objective To study the expression of CD44 gene in non-small cell lung cancer (NSCLC) tissue and the relationship with prognosis. Methods The expressions of CD44 gene in 36 specimens from 36 patients with NSCLC were determined by RT-PCR and all the patients were followed up for 3 years. Results CD44 gene was excessively expressed in 21 of 36 specimens of NSCLC tissues, excessive expression rate was 58.3%. The expression of CD44 mRNA in NSCLC tissue was related to metastasis of lymph node(χ² =9.787,P<0.01), not related to age, sex, histological types,pathological grades, TNM stages, tumor size of the patients. Kaplan-Meier survival curve showed that the survial rate of CD44-positive NSCLC tissue was lower than that in CD44-negative NSCLC tissue (χ²=5.9116, P<0.05).Conclusion The rate of lymph node metastasis is high in NSCLC patients with CD44 gene excessive expression. The detection of CD44 gene expression can guide the prognosis, radiotherapy and chemotherapy of patients with NSCLC after operation.

Objective To observe the effects of ultrasound combined with hematoporphyrin (HP) on NCI-446, PLA-80IC,PLA-801D of human lung cancer cell lines in vitro. Methods NCI-446, PLA-80IC,PLA-801D of human lung cancer cell lines which were treated with different concentrations(5,10,20 g•L^-1) of HP were used as exp erimental groups to investigate the killing role of ultrasound with HP as photosensitizer,and the cells in control group were not treated with HP and ultrosound. MTT was adopted to measure A₄₉₂.STD was used in 10 g•L^-1 Hp and NCI-446 cell lines, then the influence of HP in cell cycle and apoptotic rate was detected with flow cytom etry.Results There were significant differences of cell activity between various experimental groups and control group (P <0.05) and also between NCI-446,PLA-801C and PLA-801D(P<0.05);while no difference betwe en PLA-801C and PLA-801D. The result of flow cytometry showed that G₀/G₁ stage arrest was found in SDT group. The apoptotic rate in SDT group （19.224%±6.552%) was higher than that in control group (0.320%±0.360%) (P<0.01).Conclusion Ultrasound combined with HP has definite killing effect on human lung cancer lines NCI-446,PLA-801C and PLA-801D in vitro through inducting apoptosis.

Objective To observe the changes in contrast sensitivity(CS) and color vision in patients with primary acute angle-closure glaucoma(PACG). Methods CS and color vision tests were performed in 35 patients (35 eyes) with preclinical stage of PACG，32 patients （32 eyes） with acute attack stage of PACG and 15 normal subjects (30 eyes). Then patients were followed up 1 month (31 eyes)and 3 months(28 eyes) after operation. Results The results of CS and color vision tests were abnormal in PACG patients when they were compared with those of normal people. The threshold of CS at 18.0 c/d was increased (P<0.01) and color vision was abnormal(P<0.01) in the preclinical stage patients. The threshold of CS at 12.0 c/d and 18.0 c/d were increased (P<0.01)and color vision was abnormal (P<0.01) in the patients 15 d after operation. Conclusion Both CS and color vision are high sensitive parameters for visual function evaluating of glaucoma.

Objective To explore the clinical significance of detection of free cancer cell CK-20 mRNA in peripheral blood in patients with gastric carcinoma before and a fter operation.Methods CK-20 mRNA levels of free cancer cells in peripheral blood in 40 patients with gastric cancer and 20 cases of benign disease of stomach and duodenum before and after operation were determined with PCR technique.Results CK-20 mRNA did not express in the benign disease group. The positive expression rate of CK-20 mRNA was 22.5% (9/40) in gastric cancer group before opera tion and the positive rate was 37.5%(15/40) after operation (P<0.05). The positive rate of CK-20 mRNA after operation in group A (peripheral veins were ligated around stomach at first ) was 35%, and in group B ( peripheral veins were not ligated around stomach) was 40%, there was no significan t difference between group A and group B (P>0.05). The positive expression of CK-20 mRNA has negative correlation with degree of differentiation and Bormann typing(r=0.012，P>0.05；r=0.024，P>0.05).Conclusion There are some free cancer cells in the prepheral blood of some patients with gastric cancer before operation. Pulling and stimulating tumor in the operation may increase cancer cell dissemination. Simple ligation of peripheral veins around stomach could not completely prevent cancer cell dissemination in the peripheral blood.