To package the recombination adeno-associated virus with human U6 promoter and c-met short hairpin in order to establish foundation for research on inhibiting the expression of c-Met and cancer gene therapy. Methodsc-Met short hairpin was synthesized and linked to the down stream of U6 promoter by PCR. Adeno-associated viral vectors pSNAVUshMet(1,2) were constructed and transfected into BHK cells.Positive cell clones were selected by G418.Adeno-associated virus with U6shMet were packaged by adding rHSV1-repcap.The virus purity was analysed by SDS-PAGE and titer was assayed by dot blot.ResultsTwo fragments (U6shMet1 and U6shMet2) with c-Met short hairpin were obtained and two recombination adeno-associated virus (rAAVUshMet1 and rAAVUshMet2) with U6shMet were packaged successfully.SDS-PAGE analysis showed that the purified virus appeared clear characterized bands.The virus titer was 4×10¹²mg•L^-1. Conclusion The recombination adeno-associated virus (rAAVUshMet1 and rAAVUshMet2) with U6shMetare constructed successfully.The virus have high titer and good purity and could act as the effective vehicle of inhibiting the expression of c-Met and cancer gene therapy.

To explore the methods for isolating and culturing dendritic cells derived from rat bone marrow in vitro，and provide reference for immunological research and clinical application. Methods The bone marrow mononuclear cells(BMMNC) were obtained from bone marrow of Fischer344 rat’s limbs through density gradient centrifugation;and the mature DC was gained by BMMNC cultured in medium with recombinant rat granulocyte-macrophage colony stimulating factor (rrGM-CSF),recombinant rat interleukin-4 (rrIL-4) and recombinant rat tumor necrosis factor -α(rrTNF-α).In group 1,the rat’[KG-*3]s ovarian cancer cell line NuTu-19 freeze-melt antigens were added on the third day and the tumor cells lysates pulsed DC was acquired;in group 2,as control,nothing was added.Then the shape of DC was observed,the cell-surface phenotype and the proliferation rate were detected.ResultsThe mature DC could be induced by BMMNC co-culturing with relative cytokines,and the expressions of cell-surface phenotypes such as OX62,CD86 and MHC-Ⅱ in group 1 were higther than those in control group (P<0.01).When the ratios of stimulating cells(SC) to reaction cells(RC) were 1∶3,1∶10,1∶30  and 1∶100,the stimulating indexes(SI) in group 1 were higher than those in control group(P<0.01). Conclusion The culture method of DC from bone marrow of Fischer344 rats is established,which means the mature DC can be acquired from BMMNC.

To analyze the change of phosphotidyl serine(PS) in apheresis platelets(plts) during storage process,and to evaluate the quality of the plts during the storage period. MethodsThe apoptosis ligand Fas(CD95) was used to induce plts as apoptosis model,and by which the method to detect plts PS by flow cytometry(FCM) was established.Subsequently the resting PS expressive rate and the induced PS expressive rate of 30 apheresis plts,which induced by CD95,were determined using the FCM method established in storage process.ResultsAt the 1st,5th,7th,8th day of storage process,the PS expression of plts kept on increasing.The PS expressive rate of plts were 2.03%±0.91%,3.26%±1.86%,8.98%±4.60%,26.68%±21.28%,and 38.85%±26.43%,respectively,and there were significant differences among groups respectively(P<0.05).Under induced-operation of CD95,the PS induced-expressive rate of every storage group was obviously higher than that of 0 day group (P<0.01).The PS expression rate of plts had no difference between 7-day storage group and 0-day induced group by CD95 (P>0.05).ConclusionThe PS expression of apheresis plts increase significantly with the prolongation of storage time;Fresh plts has the ability of anti-apoptosis;Apheresis plts could keep normal function until storage 7 d.

To investigative the changes in renal function and histopathological in caulis aristolochiac manshuriensis(CAM)-induced injury in rats. MethodsMale Wistar rats were randomly divided into control group(n=10) and administration group(n=30).After CAM were administered by gavage to rats for 3,7,15 d,the blood and 24 h-urine samples were collected,24 h-urinary protein excretion,urinary retinol-binding protein (RBP),serum creatinine (Scr) and blood urea nitrgen (BUN) were examined.The pathological changes in kidney were observed at various time and compared with control group.Results① At various time after administration of CAM,the Scr,BUN and 24 h-urinary protein excretion levels were not increased compared with control group.Compared with control group,after administration of CAM for 3 d,urinary RBP content was significantly increased( P<0.01),and the values increased with the elongation of time.② The degeneration and necrosis in tubules were major pathological changes in rats after administration of CAM for 3 d，and the pathological changes were aggravation as treatment time increased. Conclusion The degeneration and necrosis in tubules are major pathological changes in rats after administration of CAM.

To clone and express functional recombination human scFv-Fc antibody against rabies virus in methylotropic yeast Pichia pastoris.MethodsThe genes of antibodies against rabies virus were amplifies from the donors vaccinatedby vaccine of PM strain produced in Vero cell line.The human recombinant scFv antibody genes were prepared by SOE PCR and inserted into yeast expression vector pPICZα carrying the human IgG Fc region.The scFv-Fc antibodies were selected from a human scFv-Fc Pichia pastoris secretory expression library.ResultsAfter three rounds of selection against the antigen of the rabies virus using ELISA assay,12 clones recognized the rabies antigen.The variable region genes of the heavy and light chains of two scFv-Fc antibodies to rabies virus were sequenced and identified as new genes.The specificity of the resulting scFv-Fc molecules for rabies antigen was established by ELISA and Western blotting analyses.It’[KG-*3]s relative molecular mass was 56 000. ConclusionThe results demonstrate that functional antibodies can be screened from the human scFv-Fc Pichia pastoris secretory expression library.

To investigate the correlation of cochlear injury and expressions of Bcl-2 and Bax in the cochlea of diabetic rats.MethodsThirty five Wistar rats were randomly divided into diabetic group(n=25) and control group(n=10).Diabetic deafness was induced in rats by intraperitoneal injection of streptozotocin,while the rats in normal group were injected with normal saline.Immunohistochemical staining was used to detect the protein levels of Bcl-2 and Bax in the cochlea after 3 months. ResultsNo significantly morphological change in cochlea of diabetic rats was found.The Bcl-2 levels in stria vascularis in diabetic group and control group were 135.32±21.69 and 45.32±9.08,respectively,there was significant difference (P<0.01).There were no changes of Bax protein expression in hair cells,spiral ganglion and stria vascularis between diabetic group and control group. ConclusionOver-expression of Bcl-2 protein in stria vascularis may be involved in the early stage of diabetic cochlear damage.

To investigate the effect of exogenous purine nucleotides on neuron nucleotide anablism depression. Methods Rat pheochromocytoma (PC12 cells) were divided into control,morphine,mono-purine nucleotide(AMP+GMP),tri-purine nucleotide(ATP+GTP),morphine+AMP+GMP and morphine+ATP+GTP groups.RT-PCR was used to examine the gene transcripts concentrations of adenosine kinase (AK) and hypoxanthine-guanine phosphoribosyltransferase (HGPRT) in each group. ResultsThe concentrations of AK and HGPRT mRNA(1.330±0.108 and 1.407±0.141 respectively) were decreased in morphine group (P<0.01,P<0.05) compared with control group(1.874±0.161 and 1.923±0.155 respectively).The expression level of AK mRNA in the group of morphine+AMP+GMP (1.626±0.171) had no difference compared with control and morphine group(P>0.05),and HGPRT mRNA expression level (1.796±0.168) was higher than that in morphine group(P<0.05).The expression level of AK mRNA in the group of morphine+ATP+GTP (1.107±0.164) was lower than that in control group(P<0.01),and HGPRT mRNA expression level (1.563±0.209) had no difference compared with control and morphine group(P>0.05). ConclusionThe exogenous supply of purine nucleotides can ameliorate the down-regulation of morphine on purine nucleotide anabolism key enzyme gene expression in PC12 cells.

To investigate the effects of adenovirus vector carrying murine interleukin-12 gene (AdCMVIL-12) on the expression of GATA-3 in mouse model with asthma. MethodsThirty BALB/c mice were randomly divided into 3 groups: control group（group A），asthma model group (group B) and AdCMVIL-12 experimental group (group C).Asthma models were established in group B and C,saline (50 μL) and 5×10⁸ PFU AdCMVIL-12 were given by intranasal administration at the 20th days.BALF were recovered at the 28th day for cellular classification.Pathological changes of bronchial and lung tissue were observed by HE staining in each groups.The expression of GATA-3 in each group was detected by immunohistochemistry and RT-PCR. Results Compared　 with group A and C,a lot of inflammatory cells were found in group B and the percent of eosinophils was the most,the expression of GATA-3 in lung tissue of group B(0.48 ±0.06) was higher than those in group C(0.16±0.07) and group A(0.18±0.04) (P<0.01). Conclusion AdCMVIL-12 can significantly inhibit airway and lung tissue inflammation in asthma model,its mechanism may be related to blocking the expression of GATA-3 in asthma model and regulating the balance of Th₁/Th₂ cytokines.

To investigate the effects of panaxadiol saponins monomer Rb₁ on action potential(AP) and L-type calcium channel in ischemic cardiomyocytes of guinea pigs. Methods The Langendorff perfusion method was used to isolatethe ventricular myocytes of guinea pigs and the ventricular myocytes were randomly divided into normal control group,ischemia group and three dose groups of Rb₁ (100,200 and 400 μm•L^-1).The AP of ventricular myocytes was recorded by current-clamp mode and the current of L-type calcium channel was recorded by voltage-clamp mode using whole clamp technique. Results Compared with ischemic cardiomyocytes group,the action potential duration(APD₅₀) and (APD₉₀) were decreased in 100,200 and 400　μm•L^-1Rb₁ groups（P<0.05 or P<0.001）,the peak current of L-type calcium channel in ischemic cardiomyocytes was decreased singnificantly（P<0.05 or P<0.01）after Rb₁ was administered.Panaxadiol saponins monomer Rb₁ inhibited the opening of calcium channel in ischemic cardiomyocytes,the effect of calcium current was reduced with the increasing of Rb₁ dose in a dose -dependent manner. Conclusion Panaxadiol saponins monomer Rb₁ can shorten AP duration of ischemic cardiomyocytes and inhibit the opening of calcium channel which may be one of the critical mechanisms of anti-myocardial ischemia of panaxadiol saponins monomer Rb₁.

To clone the rat heme oxygenase-1 (RHO-1) from rat spleen and express RHO-1 in E.coli BL-21.MethodsThe total RNA was extracted from rat spleen and amplified by reverse transcription polymerase chain reaction (RT-PCR).PCR products were cloned into pMD18-T (TA)vector followed by DNA sequencing.RHO-1 cDNA fragmentsin TA vector were subcloned into the prokaryotic expression vector pET28a(+).The recombinant pET28a(+)/RHO-1 (rRHO-1) plasmid was transformed into E.coli.The rRHO-1 was induced with IPTG and characterized by SDS-PAGE. ResultsThe cloned RHO-1 gene was composed of 870 nucleotides,and was accordance with the sequence reported in GenBank.The prokaryotic expression vector was constructed successfully.The RHO-1 protein was successfully expressed in E.coli.ConclusionThe prokaryotic expression vector of rRHO-1 has been constructed,and the fusion protein has been successfully expressed.

To investigate the changes of liver function in heroin dependent rats.MethodsUsing intraperitoneal injection with increasing doses to establish rat models of heroin administration and withdrawal.50 Wistar rats were randomly divided into five groups: control,3 d administration group,9 d administration group,3 d withdrawal group and 9 d withdrawal group.The levels of aspartate aminotransferase (AST),alanine aminotransferase (ALT) and γ-glutamyl transpeptidase (GGT) in plasma were tested.ResultsCompared with control group,the level of ALT in 3 d withdrawal group decreased significantly (P<0.01),the level of ALT in 3 d administration group decreased significantly (P<0.05),the level of ALT in 9 d administration group decreased even significantly(P<0.01),the level of ALT in 3 d withdrawal group increased apparently(P<0.01)；and the level of GGT in 9 d administration group decreased apparently. Compared with 9 d administration group,the level of AST in 3 d withdrawal group increased apparently (P<0.01),the levels of ALT both in 3 d and 9 d withdrawal groups increased apparently(P<0.01）,the level of GGT in 3 d withdrawal group increased apparently (P<0.05),and the level of GGT in 9 d withdrawal group increased even apparently (P<0.01). ConclusionThe heroin has detention and durative damage to the liver function,and can’t be eliminated immediately after drug withdrawal.

To study the inhibitory effect of antigliomatin on the expression of vascular endothelial growth factor (VEGF) in rat C6 brain glioma cells.MethodsThe rat brain glioma cells was cultivated with the stationary culture technique.The different rat C6 brain glioma cells groups were treated with 2,4,8,and 16 ng•L^-1 antigliomatin for 72 h in vitro and then the inhibitory effect of antiglioma on the expression of VEGF was observed with immunohistochemical method. Results The cytoplasm and (or) nucleus of most VEGF positive cells were stained to yellow brown.VEGF expressed significantly in cytoplasm of the rat C6 glioma cells in control group.The density of the VEGF positive cells was decreased in glioma compared with control group after treated with antigliomatin for 72 h (P<0.05，P<0.01),and the antigliomatin with differenct concentrations resulted in the different densities of VEGF,there was no difference between the group treated with 2 ng•L^-1 antigliomatin (182.3±5.7） and control group（185.6±6.5）（P<0.05）.The densities of the VEGF in the groups treated with 4,8,16 ng•L^-1 antigliomatin and CDDP(176.8±5.4,169.8±4.1,162.6±2.9,167.3±3.5）  were significantly lower than that in control group(P<0.01). ConclusionAntigliomatin has inhibitory effect on the expression of VEGF in rat C6 glioma cells.

To construct the recombinant plasmid carrying small interfering RNA(siRNA) to CXCR4 and observe dumbness effect of chemokine receptor gene CXCR4 suppression by RNA interference(RNAi),and explore the new method of more efficacious therapeutic possibilities for HIV-1. Methods Small interfering RNAs targeting CXCR4 gene were designed by using software.The complement form was obtained by annealing and interted into vector Psilencer3.1-H1neo.After sequencing,MT₄ cells were transfected using the plasmid vector.RT-PCR and flow cytometry detection were used to evaluate the suppression of CXCX4 expression in different groups.ResultsThe recombinant plasmid had the correct special fragments and DNA sequence detected by PCR,electrophoresis and DNA sequencing;The siRNA obviously suppressed the expression of CXCR4.When transfected with plasmid vector for 48 h,the inhibitory rate was 70%,compared with control and negative groups,there were significant differences （P<0.05）. Conclusion The siRNA expression vector of CXCR4 is constructed successfully.

To study the vascular endothelial growth factor (VEGF) expression and secretion of mesenchymal stem cells (MSCs) of rabbit transfected with pcDNA3-VEGF165 expression plasmid in order to construct a kind of tissue engineering artificial skull with more blood supply.MethodsBy gene reconstruction method,the VEGF165 gene was cloned into eukaryotic expression plasmid pcDNA3 and recombined eukaryotic expression plasmid pcDNA3-VEGF165 was constructed;By lipofectamine transfection method,pcDNA3-VEGF165 expression plasmid was transfected into MSCs of rabbit;By RT-PCR and Western blotting methods,the VEGF mRNA expression and VEGF protein secretion in the MSCs were detected. Results Recombined eukaryotic expression plasmid pcDNA3-VEGF165 was confirmed to be true by double enzyme digestion，two strips came to appear in 5400 bp and 600 bp of gelose electrophoresis and their sizes accorded with pcDNA3 plasmid and VEGF165 accordingly;Primarily cultured and subcultured MSCs of rabbit were successfully performed and the MSCs storehouse of rabbit was established，primary MSCs presented lymphoid form at first and then the morphology of them became circulara,polygonal or irregular forms,they were more and more like fibroblastic cells after subcultured cultivation.The expressions of VEGF mRNA and VEGF protein in the MSCs were found after transiently transfection by RT-PCR and Western blotting methods. Conclusion Recombined eukaryotic expression plasmid pcDNA3-VEGF165 can be transfected into MSCs of rabbit effectively by lipofectamine and the VEGF expression can be detected in the MSCs after transfection.

To study the inhibition of anti-PDGF on proliferation of culturedhuman retinal pigment epithelial cells (hRPE) in vitro.MethodshRPE were cultivated and were exposed to different concentrations of anti-PDGF (0,1×10^-6,5×10^-6,1×10^-5,5×10^-5 and 1×10^-4 mg•L^-1) respectively .Growth curves were measured with cell counting and the vitalities of cells were examined by percentage of vital cells and total cells.Using MTT staining colorimetric to measure the inhibitory rate.The changes of cell cycle of hRPE were collected and their growth were detected with FCM analysis and the morphological changes of cells were observed by light microscope and electron microscope. Results Anti-PDGF of 1×10^-6mg•L^-1 stimulated hRPE proliferation slightly.Anti-PDGF at dosages ranging from 5×10^-6mg•L^-1 to 1×10^-4mg•L^-1 inhibited cell proliferation effectively in a dose-dependent and time-dependent manner (P<0.05).The inhibitory rates of anti-PDGF were 12.55%,43.72%,55.1% and 55.1%.hRPE might be blocked at G₂/M phase in their growth cycles after treated with anti-PDGF (5×10^-5mg•L^-1) for 72 h,and the electron microscopy result showed a significant reduction of microvilli,margination of nuclear chromatin and intact plasmalemma. Conclusion Anti-PDGF can inhibit hRPE in a dose-dependent and time-dependent manner.It’s inhibitory function lies in inducing apoptosis without resulting in cell putrescence.

To investigate the role of TLR9 in the glomerulonephritis through observing the changes of Toll-like receptor 9 in the glomerulo- nephritis kidney tissue with or without CpG-ODN stimulation . Methods Wistar male rats were randomly divided into normal group (N),nephritis model group(M),CpG-ODN group(CpG) and GpC-ODN group(GpC).The urine samples were collected at 2,4,6 and 8 weeks after treatment,respectively.Blood samples were collected at the end of the last urine sample was collected,and the kidney tissue was collected,then the rats were killed.24 h urine protein was measured by Coomassie light blue technique.Serum album and renal function were determined by serological method.The pathologic changes of kidney were observed by light microscope and NF-κB p65 expression was detected using immunohistochemystry,RT-PCR was performed to detect the expressions of TLR9,INF-γ and IL-6 mRNA. Results The expression of TLR9 was lighter in group M,and significantly increased after CpG-ODN stimulation compared with group M.Furthermore,24 h urine protein excretion was markedly increased,serum album was markedly decreased.The histopathologic changes of kidney were more severe.The mesangial cells (MCs) proliferated diffusifully in midrange and wide range,some of the glomeruli formatted cellularity crescent,micrangium loop was limitted,mononuclear macrophile cells were seen in the mesangial region. Conclusion Inflammatory factors mediated by TLR9 can deteriorate the biochemical and histopathologic changes.The immunologic reaction mediated by TLR9 is one of the mechanisms for the glomerulonephtitis’ progression.

To investigate the effect of taurine (Tau) on the myocardial fibrosis and its mechanism. MethodsThe rat model of myocardial fibrosis was built by subcutaneous injection of isoprel(Iso).And the rats were divided into control group,model group and taurine group(80,160,320 mg•kg^-1•d ^-1).Hydroxyproline measurment was carried out for detecting collagen quantification in myocardium tissue.Histopathology changes were detected by HE staining.In vitro,the cultured neonatal rat cardiac fibroblasts(CFb) were divided into control group,model group and taurine group(40，80，160mmol•L^-1).The proliferation of CFb was induced by angiotensin Ⅱ(AngⅡ) and detected by the thiazoleblue (MTT)colorimetric assay.TGF-β₁ level was determined by ELISA technique.The expression of cyclin dependent kinase inhibitor p27 was assessed with flow cytometry. Results In vivo,the contents of hydroxyproline were decreased in Tau(160，320 mg•kg^-1•d^-1) groups as compared with model group(P<0.01 or P<0.001).HE staining results showed that myocardium fiber appeared hyperplastic and were arranged disorder and kytoplasm swelled in model group and Tau relieved the damage of myocardium induced by Iso.CFb proliferation rate and the level of TGF-β₁ were greatly declined when CFb was treated with taurine (40,80 and 160 mmol•L^-1) for 24 h (P<0.05 or P<0.01).The P27 expression was significantly increased in Tau groups（80 and 160mmol•L^-1）as compared with model group(P<0.05 or P<0.001).ConclusionTau can inhibit the proliferation of CFb and metabolism of collagen and offer protection against the myocardial fibrosis by decreasing the level of TGF-β₁ and up-regulating P27 expression.

To investigate the effect of 12-lipoxygenase(12-LOX) inhibitorinducing the apoptosis of human hepatocellular carcinoma cell line HepG2 on the expression of survivin gene. Methods HepG2 cells were cultivated in RPMI-1640 medium.12-LOX mRNA was analyzed by reverse transcription polymerase chain reaction (RT-PCR).The effect of baicalein on the proliferation of the cells was detected by thiazolyl blue tetrazoliumbromide (MTT) method.The subcellular structures were observed under electron microscope.DNA ladder pattern on agarose gel electrophores was used to evaluate the apoptotic index of hepG2 cells.The expression of survivin mRNA was analyzed by RT-PCR. Results12-LOX mRNA was expressed in human HepG2 cells.At concentrations from 20 to 80 μmol•L^-1,baicalein inhibited the proliferation of HepG2 cells in a concentration- and time-dependent manner from 24 to 72 h.There were significant differences between any other groups (P<0.05).Morphological changes such as chromatin condensatio,apoptotic bodies were observed after treated with baicalein.Significant apoptosis was also induced by baicalein,and the apoptotic index was detected by agarose gel electrophores which showed eveident DNA fragmentation.12-LOX inhibitor depressed the expression of survivin mRNA in a concentration- and time-dependent manner from 24 to 72 h. Conclusion 12-LOX inhibitor can induce the the apoptosis of human hepatocellular carcinoma cell line HepG2,and the probable mechanism is to depress the expression of anti-apoptosis gene survivin.

To investigate the effects of angiotensin Ⅰ-converting enzyme inhibitor Enalaprilat （Ena） on proliferation and nitric oxide synthase-nitric oxide system of neonatal rat cardiac fibroblasts （CFb）and to probe its anti-cardiac fibrosis mechanism.MethodsThe cultured neonatal Wistar rat CFb were divided into control group,model group and three doses of Ena groups.CFb were isolated by trypsin digestion method.MTT colorimetric assay was adopted to evaluate cell proliferation,flow cytometry was used to measure cell cycle,nitric acid reductase method and spectrophotometry were used to detect the NO contents and NOS activity respectively with Ena. Results Ena could inhibit CFb proliferation induced by AngⅡ,there were significant differences of MTT values between model group and Ena groups after treated for 24,48 and 72 h(P<0.05 or P<0.01);compared with model group,Ena increased the percentage of cells at phase G₀/G₁,and decreased the percentage of cells at phase S,there were significant differences(P<0.05 or P<0.01);compared with model group,Ena increased the level of NOs/NO of CFb supernatant,the NOS activity and NO content increased significantly with the increasing of dose and time (P<0.05 or P<0.01) . ConclusionThe anti-proliferative effects of Ena on CFb can be attributed to enhancing nitric oxide synthase-nitric oxide system of cardiac fibroblasts and accordingly increasing NO level.

To study the regulatory function of Boschniakia rossica polysaccharide on immune organ of mice bearing tumor and effect on immunological function.MethodsThe rats inoculated with hepatoma carcinoma cell H₂₂ were divided into six groups at random:50,100,200 and 400 mg• kg^-1•d^-1 Boschniakia rossica polysaccharide groups;negative group and positive group.10 d later,the mice were killed,then the spleens and thymuses were weighed,the effect of Boschniakia rossica polysaccharide on spleens and thymus index were determined.MTT was used to determine the activity of NK cells,lymphocyte transformation test was performed to determine LTT,the phagocytic activity of peritoneal macrophage was observed.The tumor inhibitory rate was calculated. Results Boschniakia rossica polysaccharide depressed the growth of tumor obviously,its tumor inhibitory rate was 38.86%.Compared with negative control group,the spleen indexes of mice bearing tumor in experiment were increased groups(P<0.01)，and thymus index had tendency of decreasing,the transformation efficiency of T lymphocyte,the activity of NK cell and the phagocytic activity of peritoneal macrophage were enhanced(P<0.01),they were positively correlated with medicative dosage.ConclusionBoschniakia rossica polysaccharide can depress the growth of tumor,and has the regulatory effects on immunological function in mice.

To study the inhibitory effects of Fe³⁺-CMC on the expressions of transforming growth factor β(TGF-β) and fibroblast growth factor (FGF) of injured parts of postoperative peritoneal. Methods Sixty Wistar rat models with abdominal adhesion were randomly divided into control group and expriment group.At the 1st,3rd,5th,7th,14th day and 2 months later,5 rats were selected respectively in two groups and killed.The adhesion tissues were gained and the expressions of TGF-β and FGF were observed using immunohistochemistry technique. Results The expressions of TGF-β and FGF in expriment group at the 3rd,5th,7th and 14th day after operation were lower than those in control group at the same time (P<0.05). ConclusionFe³⁺-CMC can inhibit the expressions of TGF-β and FGF of injured parts of postoperative peritoneal,then decrease the postoperation adhesion.

To observe the action of lowering blood fat of corn filament beverage (CFB) on hyperlipemia rats and mice.MethodsSixty rats were randomly divided into control group,alloxan model group,kanglishu capsule 0.70 g•kg^-1 group,CFB 7.00,3.50,1.75 mL•kg^-1 groups(n=10). Rats in each group were exhibited once a day for 21 d,on the seventh day,fourteenth day,twenty-first day, the contents of serum total cholesterol (TCHO) and triglyceride(TG) were determined,and liver malonaldehyde (MDA) level was measured. Another 60 mice were divided into 6 groups with the method mentioned above,mice in each group were exhibited once a day for 14 d,on the fourteenth day,the serum contents of TCHO and TG were determined,and liver MDA level was determined.Another 72 mice were randomly divided into control group,egg yolk model group,Expellant and Cleanning Fat Capsule 0.4 g•kg^-1 group,CFB 10.0,5.0,2.5 mL•kg^-1 groups (n=12).Mice in each group were exhibited once a day for 10 d.16 h before the last administration,except control group,the mice in other groups were injected with 75% yolk physiological salt solution 0.5mL through the abdominal cavity,and began to starve,1 h after the last administration, the contents of serum TCHO and TG were determined,and liver MDA level was measured. Results Compared with alloxan model group,TCHO and TG in hyperlipemia rats of CFB 7.00 mL•kg^-1 all reduced obviously　(P<0.001),the MDA content of each dose all reduced obviously(P<0.001,P<0.001,P<0.01),and showed a certain relation between quantity and effect. Compared with alloxan model mice,TCHO and TG in hyperlipemia mice of CFB 10.0 mL•kg^-1 all reduced obviously（P<0.01）,the MDA content of each dose all reduced obviously（P<0.001,P<0.001,P<0.01）,and showed a certain relation between quantity and effect. Compared with egg yolk model mice,TCHO in hyperlipemia mice of CFB 10.0 mL•kg^-1 all reduced obviously（P<0.01）,TCHO in hyperlipemia mice of CFB 10.0,5.0 mL•kg^-1 all reduced obviously（P<0.01,P<0.05）, the MDA contents of CFB 10.0,5.0 mL•kg^-1 reduced obviously （P<0.001,P<0.01 ）. CFB 7.0 mL•kg^-1was superior to Kanglishu Capsule in reducin g TCHO,TG and MDA,CFB 10.0 mL•kg^-1 was superior to Expellant and Cleanning Fat Capsule. ConclusionCFB has the function of lowering blood fat on hyperlipemia rats and mice.

To compare the efficiency of IL-18 and IL-12 in inducing the tumor specific CTL in vitro,and test the cytotoxicities of tumor specific CTL induced in the same culture system with IL-18 or IL-12. MethodsThe NK cells,T cells and dendritic cells were separated from fresh PBMNCs by using the Stem Sep^TM immunomagnetic beads.Cell phenotypes of the purified cell populations were identified with FCM technique.The cell co-culture system was established as follows:the cell preparations in which definite cell subset had been deleted was co-cultivated with mitotic-inactivated tumor cells in the presence of rhIL-18 and/or rhIL-12.Cytotoxicities of the various effector cell preparations to a series of tumor cell lines were examined by the isotope releasing assay.Results In the  cell co-culture system in vitro,rhIL-18 or rhIL-12 alone and dose-dependently induced tumor specific cytotoxicities.The level of cytolytic activity of the tumor specific CTL induced by IL-12 was substantially inferior to that induced by IL-18 (P<0.01). ConclusionThese results suggest that IL-12 does not induce tumor-specific CTL as efficiently as IL-18 does in this co-culture system under these conditions，but the synergistic effect could be observed during administration of IL-18 and rIL-12.

To establish analysis methods of two-dimensional (2-DE)gel electrophoresis for human osteosarcoma. Methods A series of methods,including Immobilized pH gradient were used as ID.Some applications,such as sample preparation used as choice of IPG gel,were improved.Coomassie brilliant blue staining,ImageMaster 2D Elite 3.01 analysis software,MALDI-TOF/ TOF MS and SWISS-PROT database serching were used to separate and indentify the proteins of human osteosarcoma. ResultsThe good use pattern including repetitive experiments showed that in three experiments,the amount of protein spots of the same team sample deviates from the relative standard as following,the average of variation coefficient (%) : 23.00±10.11 and 20.33±9.90;and the range of variation coefficient (%） were：3.80-6.89 and 2.70-6.89 from osteosarcoma and normal group respectively.The isoelectric point and molecular weigh of the same protein spots in three experiments deviated from relative standard as following:(8.93%±1.17)%，(10.16±2.02)%,(10.87±3.86)%, respectively.Therefore,better resolution and repetitive 2-DE atlas were obtained.The proteins from 11 pairs of sample were analysed by mass spectrometry,9 identified proteins(transthyretin precursor, Triosephosphate isomerase，slow skeletal muscle,cardiac muscle Troponin T,Cofilin-1，Myosin light chain 1,Calgranulin B，Heat-shock protein,Annexin A5, Fanconi anemia group D2 proteins) were more abundant in osteosarcoma tisstues and 2 proteins manganese SOD and carbonic dehydratase appeared down-regulation in osteosarcoma tisstues. Conclusion This optimized 2-DE map is an important tool for further study on osteosarcoma,and these identified proteins were related -proteins with osteosarcoma.It is suggested that the changes of the proteome are involved in the pathology processe of osteosarcoma.

To observe the expressions of glial cell line derived neurotrophic factor(GDNF) and it’[KG-*2]s receptor after transplanting mesenchymal stem cells (MSCs) to rats with middle cerebral artery occlusion (MCAO). Methods Adult Wistar rats were randomly divided into MSCs transplantation group(transplantation group),equivalence saline injection group(saline group) and sham-operation group.At the 1st,3rd,5th,7th day after transplantation,the expressions of GDNF protein,GFRα1 and c-Ret mRNA were detected by using immunohistochemical and in situ hybridization.Results The GDNF positive cells at the periphery were more than those in ischemic center,and at the 7th day after transplantation of MSCs,the expression of GDNF was higher than those in sham-operation and saline groups(P<0.05).The GFRα1 and c-Ret mRNA can be detected in the integrated neurons.At the 7th day,the expressions of GFRα1 and c-Ret mRNA in transplantation group were higher than those in sham-operation and saline groups (P<0.05). ConclusionMSCs can secrete and/or promote the nerve cells secrete GDNF,and increase the expression of GDNF receptors in neuron.MSCs may play a great role in the treatment of the central nerve system diseases.

To observe the effects of oxymatrine(OMT) on arrhythmia induced by ischemia-reperfusion and explore its mechanism. MethodsThe left arterial descending coronary artery(LAD) was ligated for 5 min followed by 10 min reperfusion in order to induce ischemia-reperfusion arrhythmia in rats．Rats were randomly divided into: model group,OMT groups with different doses (15,30,and 60 mg•kg^-1).The emergency time of ventricular premature(VP),and the duration of ventricular tachycardia(VT) and arrhythmia induced by ischemia-reperfusion were observed.The isolated rabbit papillary muscles were divided into control group,OMT groups with different doses and OMT+TEA group.The action potientials in various groups were observed. ResultsCompared with model group,OMT delayed the emergency time and decreased the duration of arrhythmia induced by ischemia-reperfusion in dose-dependent manner in rats(P<0.05，P<0.01).Compared with control group, the 10%,50% and 90% repolarization levels were shortened by OMT in isolated rabbit papillary muscles(P<0.05，P<0.01).The effects were blocked by TEA. Conclusion OMT may antagonize the arrhythmia induced by ischemia-reperfusion in rats,its mechanism may 〖JP3〗be related with shortening action potential duration (APD).

Abstract:ObjectiveTo investigate the expression of DcR3 mRNA in cells of acute leukemia(AL) and its clinical significance.MethodsThe DcR3 mRNA of bone marrow mononuclear cells (BMNC) in the patients with AL was detected by RT-PCR.There were 28 AML cases,and 18 ALL cases.27 cases of non-malignant hematopathy were used as control group.The expression level of DcR3 mRNA in BMNC was detected with RT-PCR. ResultsThe positive rate of DcR3 mRNA was 67.4％ and gene expression level was 0.56±0.09 in AL group,while they were 40.7％ and 0.39±0.19 in control group, there were significant differences（P<0.05）.The positive rate of DcR3 mRNA was 71.4％ and the gene expression level was 0.56±0.09 in AML group,while they were 61.1％ and 0.53±0.08 in ALL group,there was no significant difference(P>0.05).The positive expression level of DcR3 mRNA increased when the white blood cell count was ≥30×10⁹•L^-1.ConclusionDcR3 gene might play a role by suppressing apoptosis in leukemogenesis.

Abstract:Objective To observe the expressions of TGF-β1 and E-cadherin in human tooth germ development and explore the function and significance.Methods The mandible containing the tooth germs from 36 foetus bodys aged 8-34 weeks were gotten.SABC immunohistochemistry was used to detect the distribution of TGF-β1 and E-cadherin on human tooth germs.Results TGF-β1 and E-cadherin expressed in different stages of tooth development.During bud stage and cap stage,the TGF-β1 expression was positive in tooth bud epithelium,dental lamina,outer enamel epithelium and inner enamel epithelium.During bell stage,the TGF-β1 expression was strongly positive in inner enamel epithelium,enamel knot,stratum intermedium,dental lamina,odontoblast,ameloblast and positively expressed in the dental papilla cells which neared to odontoblast and dental sac.During bud stage and cap stage,the E-cadherin expression was positive in tooth bud epithelium and dental lamina.During early bell stage,the E-cadherin expression was positive in ameloblast and outer enamel epithelium.During middle bell stage,the E-cadherin expression was strongly positive in ameloblast,odontoblast and positively expressed in outer enamel epithelium and the dental papilla cells which neared to the odontoblast.During late bell stage,the expression of E-cadherin strongly positive in odontoblast,ameloblast which neared to the hard tissue and epithelial root sheath. Conclusion TGF-β1 is an important regulatory factor which participants in tooth g erm cell differentiation and morphous development.E-cadherin has close correlation with development,secretion and mineralization of enamel and dentin.

Abstract:Objective To study the expressions and significances of matrix metalloproteinases-9 (MMP-9) and tissue -inhibitor of matrix metalloproteinase-1 (TIMP-1) in transitional cell carcinoma of bladder (TCCB).Methods Immunohistochemistry and RT-PCR were used to detect the expressions of MMP-9 and TIMP-1 in 47 surgical respected specimens from patients with TCCB (experimental group) and 5 normal bladder tissues (control group).Results MMP-9 and TIMP-1 had different degree expressions in experimental group,but the expressions were weak or negative in control group.Furthermore,the positive expression levels of MMP-9 were significantly increased with stages and grades (P<0.01); however the expression of TIMP-1 did not show significantly correlation with different grades and stages of TCCB tissue(P>0.05).Both MMP-9 and TIMP-1 had no significant correlation with first-occurrent TCCB and recrudescent TCCB.Conclusion MMP-9 plays advance roles in the occurrence and development of TCCB; the unbalance between MMP-9 and TIMP-1 is one of important molecular mechanisms in the aggressive and metastasis biologic behavior of tumor.

Abstract:Objective To study the antileukemia immune response of IL-18 gene transfected dendritic cells(DCs) of chronic myelogeous leukemia (CML).MethodsDCs were transfected with IL-18 gene by liposomes in CML.The expression of IL-18 in IL-18 transfected DCs was detected.The percentages of CD80⁺ and CD86⁺ cells in IL-18 tranfected DCs were determined by FCM.The proliferation of T cell,NK and specific CTL kill activity induced by IL-18 gene transfected DCs were detected.ResultsThe quantity of IL-18 in IL-18 tranfected DCs was (596±34.1)pg/2×10⁶cells/48 h,while the culture medium of mock-transduced DCs and DCs did not secrete detectable levels of IL-18.The percentages of CD80⁺ and CD86⁺ cells in IL-18 tranfected DCs were higher than that in mock-transfected DCs（P<0.05）.The proliferation of T cell,NK and specific CTL kill activity induced by IL-18 gene transfected DCs were much stronger than that by mock-tranfeced DCs（P<0.05）.The activity was S/R-dependent or R/T-dependent.ConclusionThere is cooperativity of IL-18 and DCs in antitumor.

Objective To investigate the relationship between the serum C-reative protein (CRP),lipoprotein(a)［LP(a)］ fibrinogen (FG)and elderly acute cerebral infarction. Methods 106 elderly patients and 70 healthy elderly persons as control were enrolled in this study.The serum CRP,LP(a) and FG levels were determined with immunoturbidimetry and compared among various severe degree and infarct size groups. Results ①Compared with control group,the serum CRP,LP(a) and FG levels in elderly acute cerebral infarction group increased significantly;they were (14.06±2.17) mg•L^-1,(322.34±6.31) mg•L^-1and (8.24±2.62) g•L^-1,resp ectively(P<0.05).② The serum CRP,LP(a)and FG levels were different among various infarct size groups,large infarct size group>small infarct size group>lacunar infarction group (P<0.05 or P<0.01).③The serum CRP,LP(a),FG leve ls were different among various severe degree groups,serious group>midrange group>mild group (P<0.05 or P<0.01).Conclusion The relationship between the serum CRP,LP(a) and FG levels and degree of acute cerebral infarction in elderly patients is intimate.The higher the serum CRP,LP(a) and FG levels were,the larger the infarct size and the more severe the pathogenetic condition.So the serum CRP,LP(a) and FG levels are risk factors for acute cerebral infarction and used to evaluate the severity and infarct size of acute cerebral infarction in elderly patients.

Objective To study the effect of ketamine and propofol on levels of perioperative plasma cytokines in gastric cancer patients underwent radical gastrectomy.Methods Forty ASAⅠ-Ⅱ patients with gastric cancer were randomly divided into ketamin group(Kt group,n=20)and propofol(Pf group,n=20),peripheral vennes blood samples were taken before anesthesia,1 h after skin incision,at the end of surgery and 24 h after operation.The plasma TNF-α,IL-6,IL-8 and IL-10 levels were determined respectively with ELISA methods.Results The levels of plasma TNF-α,IL-6 and IL-8 at 1 h after incision,at the end of surgery,and 24 h after operation in Kt group were increased,but there were no significant differences compared with before anesthesia(P>0.05).In Pf group,the levels of plasma TNF-α,IL-6 and IL-8 at 1 h after skin incision and the end of surgery were higher than those before anesthesia and those in Kt group （P<0.05 or P<0.01）.In Kt group,the level of plasma IL-10 was increased,but there was no significant difference compared with before anesthesia（P>0.05）.The plasma IL-10 level in Pf group was increased obviously compared with before anesthesia（P<0.05）.Conclusion Ketamine and propofol anesthesia can lead to take proinflammatory and antinflammatory cytokines in gastric cancer patients underwent radical gastrectomy,and keep balance between of them.Ketamine had more obviously antiflammatory effect than propofol.

Objective To investigate the correlation between the nonstructural protein 3（NS3）of chronic genotype 1b hepatitis C virus（HCV） and development of hepatocellular carcinoma (HCC). MethodsThe sera were collected from HCV-carriers and HCV-infected Chinese patients with and without HCC,HCV-RNA extracted from these serum samples were amplified by nested polymerase chain reaction (nested-PCR) to obtain the genotype 1b and NS3 region PCR products,GENETYX-Mac ver 10.1 software was used to analysis the nucleotide sequence,amino acid sequence and the secondary structure of NS3_1027-1206.Results The genotype 1b became predominant in all the patients,and the percentage had no statistically significant difference (P>0.05).The homology of nucleotide and amino acid sequence was 95%,there was no particular mutation in the NS3_1027-1206 region amino acid sequence.On the basis of secondary structure of NS3_1027-1206,the HCV isolates were classified into two types:type A and type B;type A was predominant in the carriers and non-HCC patients,type B was predominant in the HCC patients.The difference in the distribution pattern of those types between carriers and non-HCC patients was no statistically significant (P>0.05),but was statistically significant（P<0.01）between carriers,non-HCC patients and HCC patients.Conclusion There is difference in the secondary structure of NS3_1027-1206of carriers,non-HCC and HCC patients,and HCV-NS3 has correlation with the development of HCC.

Objective To study the influences of long-term basketball training on bond mineral density(BMD)at different parts of hoopman in college.Methods Fifteen male basketball athletes (training for 10 years) from Institute of Physical Education were selected as training group,fifteen non-sports male college students (with the same age )were used as non-training group.The BMD of both left and right femur of different parts of femurs(neck and ward)，the spine and the L₂,L₃,L₄ of spine and both left and right forearm and the levels of serum testosterone (T),cortisone (COR),parathyroid hormone (PTH),and bone da protein (BGP) were determined by dual energy X-ray absorptiometry (DXA) of whole body and irradiation immunity of serum.Results The peak values of BMD of L₂,L₃,L₄，right forearm,neck of left and right hip joint in training group were significantly higher than those in non-training group (P<0.01 or P<0.05).The levels of serum T,CDR,PTH and BGP at rest had no significant differences between two groups(P>0.05).Conclusion Long-time basketball training can effectively increase the BMD of L₂,L₃,L₄ of spine,right forearm,left and right hip joint.But it has no effect on the serum T,COR,PTH,and BGP at rest.

Objective To evaluate the oxidative and antioxidative levels in blood of patients with Eales disease in order to assess the relationship between the oxidantstress and Eales disease.Methods Twenty patients with Eales disease were enrolled in this study.Ten of them which were untreated before the study served as untreated group and the others which were clinical cured served as cured group.Ten healthy young blood donors were selected as control group.8-OHdG and TBARS were served as biomarker of oxidative damage to DNA and lipid peroxidation,and were detected by GC/MS and TBARS assay kit respectively.The capacity of total antioxidant,SOD,GSH-PX and the level of vitamine E in the serum were served as the marker of the antioxidant capacity,and they were detected by the corresponding assay kits.Results Compared with control group,the levels of 8-OHdG and MDA increased significantly in both groups of the patients with Eales disease（P<0.001）. And the levels of 8-OHdG and MDA in untreated group was significantly higher than those in clinical cured group（P<0.001）.The antioxidant capacity of the patients was significantly lower than that in control group（P<0.05）,and the antioxidant capacity in clinical cured group was significantly higher than that in untreated group （P<0.05）.Conclusion The oxidant stress may play an important role in etiology and pathology of Eales disease.Antioxidant therapy might be beneficial to patients with Eales disease.