Abstract:Objective To purify and assay the recombinant prostate-specific antigen (rPSA) to provide key material for study of PSA biology character,clinic detection and therapeutic application. Methods The rPSA was purified by cation-exchange chromatography from the yeast cell culture supernatant,then rPSA specificity was analyzed by Western blotting,and enzyme activity was assayed using s-2586 substrate. Results The most suitable methanol concentration to induce expression was 1.5%.The rPSA was purified by cation-exchange chromatography.The rPSA had chymotrypsin-like activity of PSA.Under different conditions enzyme activity was directly related with time.The most suitable temperature of enzyme activity was 30℃ within 3.5 h.The chymotrypsin-like activity of rPSA was obviously higher than that of PSA in seminal vesicle fluid of normal male. Conclusion The active,mature rPSA has been successfully purified.

Abstract:Objective To construct and identify the recombinant retroviral vectors expressing siRNA for MDR1 gene knock-down,sequentially to detect the reversing effect of RNAi on the multidrug resistant phenotype in MCF-7/ADR cells. Methods The target sequences of siRNAs for MDR1 knock-down were designed by IDT dna online. The DNA sequences containing a palindrome structure were synthesized and annealed to form double-stands which were inserted into linear pSUPER vectors later. The recombinant vectors were identified by agarose gel electrophoresis after cleaving of double enzymes and by sequencing,then were transfected into 293T packaging cells which produced retroviral particles in supernatant,the retrovirus soup was used to infect MCF-7/ADR cells. The expression level of MDR1 was detected from both mRNA level and protein level by RT-PCR and Western blotting,respectively. Results The recombinant pSUPER vectors expressing siRNA were confirmed by agarose gel electrophoresis after cleaving of double enzymes and by sequence analysis. The inserted oligonucleatide sequences were identical to the original sequences and in corresponding position. Compared with control group,the inhibitory effects of pSUPER.retro-MDR1 and pSUPER.retro-MDR2 were significant. Conclusion pSUPER recombinant retroviral vectors expressing siRNA for MDR1 knock-down is construted successfully,and it has inhibitory effect on the expression of MDR1 gene in MCF-7/ADR cells.

To prepare anti-activin receptor-interacting protein 1 (ARI P1) polyclonal antibody.Methods The GST-ARIP1 fusion protein was expressed in E.coli BL21.A polyclonal antibody against ARIP1 was obtained by immunizing a rabbit with the purified GST-ARIP1 and the localization of mature ARIP1 protein in mouse brain tissues was detected by immunohistochemistry using the prepared anti-ARIP1 antibody. Results Anti-ARIP1 polyclonal antibody could bind specifically with recombinant ARIP1,but not recombinant ARIP2.Immunohistochemical staining showed that ARIP1 mainly expressed in hypothalamus and hippocampus using the prepared anti-ARIP1 antibody. Conclusion The polyclonal antibody against ARIP1 has been successfully prepared and can be used to do immunohistochemical analysis for ARIP1 protein expression.

Abstract:Objective To make a comprehensive evaluation of nano-composite of poly(L-lactide) and surface grafted hydroxyapatite(PLLA/PLLA-gHA) as a new material. Methods According to the evaluated critera of medical implanted materials biology and animal trial recommended in GB/T 16886 and IS0 10993 criterion,the new material was carried out on acute systemic toxicity test,haemolysis test,muscular implantation test and subcutaneous injection test.The extract liquid of new material was injected into mice by vena caudalis to test common station,toxic reaction of it at different time,the results were used to evaluate the acute systemic toxicity.Fresh anticoagulant cony blood was mixed with extract liquid of new material with density of 100 g•L^-1 to measure each absorbance with spectrophotometer and work out the corresponding rate of haemolysis.The red punctuation and hydropsia of rabbits were observed at different time by subcuntaneous injecting extract liquid into the back of rabbits.PLLA/PLLA-gHA composite plates were implanted into the sacrospinal muscle of rabbits.Cony venous blood was extracted to detect indicatrix of hematology at diferrent time.The material and surrounded tissues were taken out from animals at the 14th,30th,60th,90th,180th,360th day to examine anatomic and pathological changes. Results Rabbits with PLLA/PLLA-gHA composite had good general condition.There was no any acute systemic toxicity in vivo.Data of AST and Scr had no significant difference between experimental group and control group.The hemolysis rate of extrac liquid was 1.22%,which was under the standard criteria(5%).No red punctuation and light hydropsia were observed at different time in the subcutaneous injection test.The inflammation cytochange of PLLA/PLLA-gHA composite group was similar with that of control group in early days,which was met with the general regularity of inflammatory outcome.The fibrosis membrane surrounding the PLLA/PLLA-gHA composite became thinner gradually with the elongation of implantation time.The fibrosis membrane grew into the material at the 360th day.The degree of the fibrosis membrane was below class Ⅰ. Conclusion The new absorbable type PLLA/PLLA-gHA composite has excellent biocompatibility and security.

Abstract:Objective To study the emmetropization in guinea pig eyes during the normal development from birth in order to provide theoretical basis to use guinea pigs as model for research on near-sightedness. Methods Sixty-four guinea pigs were assigned to 8 groups (n=8,4 male/4 female).Each group underwent a series of ocular examinations at one of the 8 time-points (0,1,2,3,5,7,9 and 11 weeks), including refraction(R),radius of corneal curvature (RCC),depth of anterior segment(AS),thickness of crystalline lens (CL),length of vitreous chamber (VC)and axial length (AL).Pooled results from both eyes of the same animals with mixed sexes can be used for further assessment of the emmetropization with the associated biometrical changes of the eye.Correlations were made between RCC,AS,CL,VC,AL and R. Results There were no significant differences between the right eye and left eye or between male and female in results of all the examinations. The refraction at birth was (+5.25±0.22 )D　 in guinea pigs.This value rapidly decreased during the first 3 weeks,approaching(+1.34±0.61)　 D by 11 weeks. There was no significant difference in refraction between 9 and 11 weeks (Ｐ= 0.21 5). The RCC was (3.23±0.01) mm at birth,AS was (1.20±0.00) mm at birth,CL was (2.72±0.02) mm at birth and VC was (3.27±0.01) mm at birth,they increased within the first 3 weeks despite a transient decrease in the RCC within the first week.Such an increase continued except the AS which became constant after 3 weeks.The VC was more correlated to the emmetropization (r=－0.818，P<0.01) than the other 3 components (r=－0.558 to －0.680，P<0.01 ). Conclusion In guinea pigs,the end point of emmetropization is among 9 to 11 weeks,the emmetropization process in guinea pigs is mainly related to the increase in the vitreous chamber lenghth.

Abstract:Objective To observe the dose-rate effect of adaptive response of apoptosis and cell cycle progression induced by low-dose ionizing radiation in EL-4 lymphoma cells in vitro in order to reveal the possible mechanism of biological effect and adaptive response induced by low dose radiation. Methods The experiment was divided into D2 (challenging dose),D1 (inductive dose) + D2 and sham-irradiation groups.EL-4 lymphoma cells were irradiated with D1 (75 mGy,6.25－200.00 mGy•min^-1) and D2 (1.5 Gy,287 mGy•min^-1),the time interval between D1 and D2 was 6 h. The percentage of apoptosis and each cell cycle phase were measured with flow cytometry.Results When the dose rates of D1 were 6.25－50.00 mGy•min^-1,the percentages of apoptosis in the D1 + D2 group were significantly lower than those in the D2 group (P<0.05);meantime,the percentages of G₀/G₁ phase cells decreased significantly (P<0.01),and the percentages of S phase cells increased at different degrees. Conclusion When the dose rates of D1 (75 mGy) are 6.25－50.00 mGy•min^-1,D2 is 1.5 Gy (287 mGy•min^-1),and the time interval between D1 and D2 is 6 h,the adaptive response of apoptosis and cell cycle progression in EL-4 lymphoma cells in vitro could be induced.

Abstract:Objective To explore the effect of low dose radiation(LDR) on biological characteristics of human mesenchymal stem cells from bone marrow (BM-MSC). Methods The P4,P5,BM-MSC were exposed to X rays at the dose of 50,75,and 100 mGy (dose rate 12.5 mGy•min^-1). The cell growth,cell cycle and apoptosis of BM-MSC treated with LDR were determined. Results Compared iwth control group,the cell growth rates of BM-MSC treated with LDR(50,75,100 mGy) were obviously increased from the fifth day(P<0.05).Compared with control group at the same time,the percentage of G₀/G₁ stage cells gradully decreased (P<0.05),and after treated for 72 h,the growth rate was the lowest. However,the percentage of S stage cells e gradually increased after treated for 48 h and 72 h(P<0.05). The most one was 75mGy group at 72 h(68.88%). The percentages of apoptotic cells had increasing tendency at 24 h and 48 h in all dose groups,while had decreasing tendency at 72 h. Conclusion LDR has hormesis effect on BM-MSC.

To observe the effect of rhKD/APP on inflammatory cytokines and ultrastructrue in experimental acute necrosis pancreatitis(ANP) in rats.Methods ANP model was induced by injections of 3.5 % sodium taurocholate into the main pancreaticduct of rats.Sixty rats were randomly divided into 6 groups（n=10）:sham group;model group;low,mediate,high dose rhKD/APP groups(4×10⁴ ,8×104 and 16×10⁴ kU•kg^-1）；aproitin group.TNF-α and Ｎ activity wereassayed and ultrastructrue changes were observed after treatment with rhKD/APP.Results Compared with sham group,the levels of TNF-α and NO in serum in model group rapdily raised(P<0.01).Compared with model group,the levels of serum TNF-α and NO in mediate and high dose rhKD/APP groups were decreased(P<0.05 or P<0.01);but there was no significant difference compared with aproitin group.The ultrastructure results of pancreas tissue showed that there were a lot of microvilli on cell surface,the nucleoli were significant,and secretory granules inreased. Conclusion rhKD/APP can reduce the inflammatory response of pancreas,and has the rapeutic effect on rats with ANP.

To research the stress distribution in cervical bone of Perfect implant and Replace implant in delayed loading.Methods The three-dimensional finite element（FEM）models of the maxilla anterior part were established by CT scanning,video transportation,transformation technology,three-dimensional finite element（FEM）approach,then the stress distribution at alveolar crest in FEM models of Perfect implant and Replace implant was analyzed. Results When Perfect implant was placed in bone and delayedly loaded,the Von Mises stress and compressive stress of alveolar crest were less than Replace implant in the same condition,while the tensile stress was more. Conclusion The stress distribution of cortical alveolar ridge in Perfect implant with delayed-loading is similar to that in Replace implant.Considering these results invitodynamics exclusively,Perfect implant won’[KG-*3]t accelerate the bone resorption speed at cervical alveolar ridge.

To study the therapeutic effect of attenuated salmonella typhimurium with siRNA-Stat3 plasmid on orthotopic transplantation tumor model of liver in C57BL/6 mice. Methods The siRNA-Stat3 plasmid was transfected into attenuated salmonella typhimurium after that orthotopic transplantation tumor model of liver in mice were infused with attenuated salmonella typhimurium and then the gene expression and function were observed.Results Compared with mock group and pGC-Si-Scramble group,the growth of orthotopic transplantation tumors of liver was inhibited significantly in siRNA-Stat3 plasmid group(P<0.05),the tumor weight was reduced obviously(P<0.05),the expression levels of protein and mRNA were down-regulated (P<0.01).Conclusion The　 attenuated salmonella typhimurium which carrying siRNA-Stat3 plasmid has the inhibitory effect on orthotopic transplantation tumor of liver.

To explore the method of secretory expression of recombinant human oncostatin M (rhOSM) in Pichia pastoris. Methods The human embryonic genomic DNA was used as templet to obtain the sequence of mature hOSM gene by PCR, the expression vector of Pichia pastoris of pPICZαC-hOSM was constructed. The recombinant plasmid was transformed into Pichia pastoris X-33 via electroporation.The transformed positive strains were screened by PCR. The supernatant were analyzed by SDS-PAGE and Western blotting to screen Pichia pastoris engineer bacteria with highly effective expression of rhOSM. Results The mature hOSM DNA was obtained by PCR. SDS-PAGE and Western blotting analysis showed there was rhOSM in the culture supernatant induced by methanol with 28 000 of molecular weight. Conclusion Pichia pastoris can express rhOSM efficiently, the expression level of rhOSM reaches at 45 mg•L^-1 in flask scale.

To explore the adverse effects of ultraviolet B(UV-B) on the skin of guinea pig.Methods Guinea　 pig skin was irradiated with UV-B,the skinchanges in external appearance,pathology,and the contents of OH and O₂^- produced in the skin were determined to study the adverse effects of UV-B on the guinea pig skin.Results UV-B caused red swelling and desquamation of skin,with the increasing of the UV-B irradiation,the cells in stratum spinosum began to proliferate vigorously,the MDA and ROS contents in UVB radiation group were significantly higher than those in control group(P<0.05). Conclusion UV-B can cause injury to guinea pig skin and has the potential to produce skin cancer.

To study the effect of hmSD on apoptosis of PC3 cells induced by NaBT and its mechanism.Methods PC3 cells and PC3-hmSD stablely transfected with hmSD were used as target cells.Four groups were set up:control,NaBT 2.5 mmol•L^-1,NaBT 5.0 mmol•L^-1 and NaBT 10.0 mmol•L^-1.The survival rate of cells was detected by MTT assay,apoptotic rate was determined with AO/EB assay,intracellular ROS was examined with DCFH-DA staining,the expressions of Bcl-XL,P65 and Caspase 9 were analyzed by Western blot ting method. Results ①The survival rates of PC3-hmSD cells treated with NaBT for 48 and 72h were higher than that of PC3 cells（P<0.05）. ② The apoptotic rate of PC3-hmSD cells treated with NaBT 5.0 mmol•L^-1 for　 4 h was lower than that of PC3 cells（P<0.05）.③ There was no difference of ROS between both cell lines treated with NaBT.④ The result of Western blotting showed that the expression of Bcl-XL decreased in NaBT-treated PC3 cells,but it increased in PC3-hmSD cells.Compared with control group,the expressions of P65 and Caspase 9 increased in NaBT-treated cells,but the expression of P65 increased and the expression of Caspase 9 decreased in hmSD overexpressed cells（P<0.05）. Conclusion ① NaBT can induce the apoptosis of PC3 cells,and hmSD can reverse it.② The pathway of hmSD against apoptosis may be no relation with the suppressed ROS synthesis.③ The antiapoptotic mechanism of hmSD may be involved in down-regulation of Caspase 9 and up-regulation of P65 and Bcl-XL expressions.

To investigate the effect of citicoline on dopaminergic neuron injury induced by MPP⁺. Methods E14 mouse embryos were used to make mecencephalic dissociated culture.10 μmol•L^-1 MPP⁺ was used to set up the cell model of damage of dopaminergic neurons.The cell cultures were divided into control group,MPP⁺- trerated group,and another five citicoline (with different concentrations of 0.01、0.10、1.00、10.00 and 100.00 μmol•L^-1,respectively) and MPP⁺ -cotreated groups. Tyrosine hydrolysis (TH) immunochemical stainning was used to identify dopaminergic neurons.The morphological changes and the number of dopaminergic neurons were evaluated. Results On the 12th day in vitro(DIV),the number of TH-ir (TH immunal reactive) neurons was significantly reduced by MPP⁺ (P<0.05).The number of TH-ir neurons increased by 11.83% and 21.26% of control level when treated with 0.10 or 100.00 μmol•L^-1 of citicoline for 6 d respectively,compared with MPP⁺-treated alone group (P<0.05).The average length of the longest denrite of dopaminergic neurons was much longer in 0.01 μmol•L^-1and 100.00 μmol•L^-1 citicoline cotreated group,compared with MPP⁺-treated alone group(P<0.05).Conclusion Citicoline could effectively prevent the death and morphological changes of dopaminergic neurons induced by MPP⁺,and provide a new treatment way for Parkinson’s dis ease (PD.

To construct a eukaryotic expression vector of decorin(DCN),and observe its expression in CHO cells,in order to provide a basis for further study on the anti-tumor effect of DCN. Methods DCN cDNA was amplified by PCR.The human full-length DCN cDNA ligated into pBluescript was used as template.The fragment was ligated to the expression vector pCDNA3 previously digested with XbaⅠ and EcoR Ⅰ.The ligation mixture was transformed into competent E.coli JM109 cells.Transformants containing inserts were confirmed by restrictive digestion and DNA sequencing.The expression vector was transfected into CHO cells using lipofectamine,and transfected cells were cultivated in DMEM containing G418 (800 mg•L^-1) for about 2 months.Immunohistochemistry method was used to detect the expression of DCN protein in stably transfected cells.Results The　 PCR product was about 1 000 bp.The recombinant expression vector was identi fied by restrictive digestion and DNA sequencing. DCN protein was detectable in stablely transfected cells. Conclusion The recombinant eukaryotic expression vector pCDNA-DEC is constructed successfully and stablely transfected CHO cells are established.

Abstract:Objective To evaluate the effects of different loading occasions of orthodontic force on the stability of miniscrew as an anchorage.Methods The titanium miniscrews were respectively implanted into canine and posterior region of both upper and lower jaws of 3 crossbred dogs.The implants were immediately loaded with 200 g orthodontic force or loaded after 2 weeks of implantation.The dogs were sacrificed after 8 weeks.The samples including the microscrew implant and its around bone tissue were extracted,fixed and X-ray photographed.Then the specimens were decalcified,embedded,sectioned and stained with HE and GOMORI.The sections were examined under light microscope. Results The stability of miniscrew in posterior region of jaws was higher than that in canine region in X-ray photograph.There was more bone trabecular formation between miniscrew and jaw bone in non-immediately loaded group than those in immediately loaded group with HE and GOMORI. Conclusion The non-immediately loaded implant as an anchorage can afford more stability than the immediately loaded implant.

To construct the DNA vaccine coexpression vector containing M2 gene of influenza A virus and GM-CSF gene,which provides a basis for exploring broad-spectrum vaccine of influenza A virus.Methods Analogous　 strain of influenza A virus（H1N1） was inoculated by chicken and alantoic fluid was collected,the total RNA of influenza A virus was extracted,M2 gene was amplified by RT-PCR.Internal ribosome entry site(IRES) gene of minute virus was inserted into the multiple cloning sites(MCS) of the plasmid pVAXⅠ,then M2 gene and GM-CSF gene were in turns cloned into the MCS of the advanced and backward MCS of IRES for constructing pMIG-the coexpression vector of influenza A virus and sequencing after enzymetomy identification.Then the recombinant plasmid pMIG was transfected into COS7 cell by liposome method and the expression of the target protein was detected. Results M2 gene(300 bp),IRES gene(580 bp) and GM-CSF gene(400 bp) were successfully amplified,the result of enzymetomy identification indicated that the coexpression vector pMIG of influenza A virus was constructed,Western blotting testified that M2 protein of influenza A virus was expressed. Conclusion The coexpression vector pMIG of influenza A virus is successfully constructed.

To investigate the inhibition of proliferation and induction of apoptosis by proteasome inhibitor MG-132 on C6 glioma cell in vitro. Methods Rat glioma C6 cells were cultured with different concentrations of proteasome inhibitor MG-132 (10,20 and 50 μmol•L^-1).Cell viability was determined by MTT assay at different cultured periods.Flow cytometry was used to detect apoptosis change and HE and AO/EB staining and electronic microscope were used to detect the morphological changes of apoptotic cells.Results Compared with normal control,MG-132 significantly reduced the viability of C6 cells in 10,20 and 50 μmol•L^-1MG-132 groups (P<0.01).Moreover,the inhibitory effect was higher on high concentration MG-132 compared with low dose MG-132. After treatment with 10 μmol•L^-1MG-132 for 24 h,the apoptosis characteristic C6 cells were detected by AO/EB and HE staining.Apoptotic sub-G was detected by flow cytometry in C6 cells incubated with 10 μmol•L^-1 MG-132 for 12 h and displayed a time-dependent manner,the apoptotic percentages in 10,20 and 50 μmol•L^-1 MG-132 groups were significantly higher than that in control group (P<0.01). Conclusion Proteasome inhibitor MG-132 can inhibit C6 cell proliferation and induce C6 cell apoptosis.

To study the effects of injecting recombinant human granulocyte colony stimulating factor (rhG-CSF) and basic fibroblast growth factor (bFGF) alone or combined on actue myocardial infarction(AMI).Methods AMI models were induced by ligation of the left anterior descending artery.The survived rats were divided into four groups randomly：AMI group (MI),rhG-CSF group (G),bFGF group (B),combined group (GB).Respectively,saline,rhG-CSF,bFGF,and rhG-CSF plus bFGF were injected intraperitoneally 24 h after AMI.Also,sham-operated group (S) was established with only chest-opeaned,without ligation,and no drugs intervention. The white blood cells (WBC) and mononuclear cells (MNC) proportion in peripheral blood were counted 1 week before and 1 week after the intervention,and the number of CD34⁺ cells was observed with immunohistochemical staining 1 week after AMI in order to compare the situation of mobilization in peripheral blood;the capillary density was evaluated by HE staining both 1 and 4 weeks after AMI;their cardiac fuction was determined in vivo,the infarction size in each group was calculated,and the pathological changes in rat myocardium were observed by HE s taining 4 weeks after AMI.Results Compared with MI group,the number of WBC and MNC% in peripheral blood 1 week after AMI in G,B and GB groups were higher（P<0.01）; LVSP,+dp/dt_max and －dp/dt_max in vivo in G,B and GB groups increased further（P<0.01）and LVDP decreased lower（P<0.01）. Conclusion Injecting rhG-CSF intraperitoneally can mobilize bone marrow stem cells into peripheral blood in infarct rats.The mobilized stem cells may enter into the infarct area and induce the regeneration of cardiac-like cells and capillary endothelial cells.Injecting rhG-CSF or bFGF intraperitoneally may also promote the regeneration of capillary in infarct area.Both of them can improve the cardiac function significantly.rhG-CSF combined with bFGF would be better than use alone.

To construct the expression plasmid of porcine leukemia inhibitory factor(LIF) in the prokaryotic system,and purify the expressed recombinant porcine LIF protein as antigen to immune rabbit for preparation of rabbit anti-pig LIF polyclonal antibody,and immunolocalize of LIF in pigs.Methods The cDNA fragment encoding mature porcine LIF protein was amplified by PCR,then cloned into the prokaryotic expression vector PET-31b at restriction sites NdeⅠand XhoⅠ.After transformed into E.coli BL21 for expression,the recombinant porcine LIF protein was purifed for preparation of anti-pig LIF polyclonal antibody as immunogen.Western blotting and ELISA were used to test the specificity and titer of the purifed IgG.Immunostaining was used to assay the expression of LIF in pigs. Results The constructed expression plasmid was identified by DNA sequencing and restriction enzyme digestion.The expression of the recombinant porcine LIF protein was performed exactly in E.coli BL21.By immuning the rabbit,the polyclonal antibody was successfully prepared.The results of ELISA and Western blotting showed that the antibody had high titer(1∶10000) and specificity.By immunostaining,LIF protein was faintly expressed in porcine myocardial cell,splenic cord and monolayer covering epithelium cell and pulmonary fibroblast.Conclusion The prepared polyclonal antibody has high titer and specificity;LIF has faint positive immunoreactivity in the normal porcine,myocardial cell,splenic cord and monolayer covering epithelium cell and pulmonary fibroblast.

To study the effect of dendritic cells(DC) stimulated with C5b-9 on allogenic lymphocytes. Methods DC was stimulated with C5b-9.CD4⁺ T and CD8⁺ T cells were isolated by magnetic-activated cell sorting (MACS),and were cocultivated with DC.The maturation markers and cytokine secretion of T cells were analyzed by flow cytometry and ELISA. Results The high pure CD4⁺ T and CD8⁺ T cells were separated successfully.After cocultivated with DC,the muturation markers CD25 and CD69 of CD4⁺ T were up-regulated by 26.31% and 52.73%,and IL-2 and IFN-γ of CD4⁺ T cells were promoted from 126.3 ng•L^-1 and 156.7 ng•L^-1 to 409.2 ng•L^-1 and 471.5 ng•L^-1;the lev els of IL-10 before and after coculture were 104.3 ng•L^-1 and 107.1 ng•L^-1.However these markers and cytokine secretion of 〖JP2〗CD8⁺ T were not influenced.Conclusion C5b-9 can adjust the function of DC and regulate the immuological response of T cells.

Abstract:Objective To explore the effect of boiled fenugreek seeds on renal matrix metalloproteinase-2(MMP-2) activity in diabetic rats. Methods The model of diabetes was built with STZ in rats.The model rats were randomly divided into diabetes control groups (DM ) （n=10） and fenugreek seeds groups(FN) （n=10,and while normal control group (N) （n=10）rats was used.Diabetic rats were treated with fenugreek seeds for 12 weeks,the renal morphology and MMP-2 activity were observed in three groups . Results After diabetic rats were treated with fenugreek seeds for 12 weeks，optical microscopic examination indicated that the glomerular structure in N group was normal,the glomerular lesions in rats of DM groups were seriously and the pathologic changes of glomerular in rats of FN groups were alleviated significantly.Immunohistochemical results showed that the Col Ⅳ expression in glomerular ECM was increased in DM group compared with N group,and was decreased in FN group.The activity of MMP-2 was increased in FN group (1.41±0.18) compared with DM group (1.05±0.19) (P<0.05) and the alterations were not manif ested compared with N group (1.53±0.25).Conclusion Fenugreek seeds may alleviate renal lesions through up-regulating MMP-2 activity.

To observe the effects of 3,4,5-trihydroxybenzoic acid dimmer (TAD) in different doses on apoptosis and cell cycle progression of liver cancer SMMC-7721 cells in vitro and explore its possible mechanism of inhibiting tumor growth. Methods The experiment was divided into 0,3.125,6.250,12.500 and 25.000 μg TAD groups. Apoptosis was observed with JEM-1200EX transmission electron microscope (TEM).The changes of apoptosis and cell cycle progression were measured with flow cytometry. Results The typical features of apoptosis were found in tumor cells,the nuclei were broken to pieces,mitochondrion cristae were disrupted and vacuoles were showed in nucleus and cytoplasm in 25.000 μg TAD group observed under TEM.Meantime,the apoptotic percentages of the cells treated with 3.125—25.000 μg TAD were increased significantly as compared with control group (P< 0.001).And the percentages of G₀/G₁ phase cells treated with TAD were increased, it was significantly higher in 25.000 μg TAD group than that in control group(P< 0.001);while the percentages of S phase cells treated with TAD were decreased，especially in 25.000 μg TAD group as compared with control group(P< 0.05). Conclusion TAD could induce liver cancer SMMC-7721 cell apoptosis and G₁ phase arrest.The inhibitory effect of TAD on tumor growth could be due to apoptosis caused by G₁ phase arrest.

To study the effects of ShaJi on the indexes of oxygen metabolism such as coronary blood flow in myocardium of anesthetized thoraco-opened dogs. Methods Dogs were randomly divided into control group,4 and　 16 mg•kg^-1 ShaJi groups,and positive control group(n=6).The anesthetized thoraco-opened dog models were set up.The administration of intravenous injection was used by femoral vein.The blood pressure,heart rate and coronary blood flow(CBF) were measured.Coronary resistance,myocardial oxygen uptake rate,myocardial oxygen consumption index and myocardial oxygen consumption were calculated. Results Compared with control group,the CBF was increased (P<0.05 or P<0.01),the coronary resistance was reduced (P<0.05 or P<0.01) and the myocardial oxygen uptake rate was decreased in 4 and 16 mg•kg^-1 ShaJi groups (P<0.05 or P<0.01). But ShaJi had no noticeable effects on myocardial oxygen consumption index and myocardial oxygen consumption compared with control (P>0.05). Conclusion ShaJi can significantly ameliorate oxygen metabolism in myocardium of anesthetized thoraco-opened dogs.

Abstract:Objective To study the effects of vancomycin-impregnated polymethylmethacrylate (VCM-PMMA) on mechanics alteration and delayed release in order to guide clinical application.Methods The specimens of bone cement (each of 40 g) were divided into 7 groups depending on mixed different contents with vancomycin，containing 0，0.5,1.0,1.5,2.0,2.5 and 3.0 g vancomycin.The compressive strength,bending strength and bending elastic moduls were measured respectively.The elution examination of vancomycin from bone cement was performed by chromatography.Results There were no differences of compressive strength before elution of bone cement that were included from 0 to 3.0 g vancomycin.When the vancomycin in the content of the bone cement attained 3.0 g,the compressive strength after elution and bending elastic were lower than those in control group（P<0.05）.Bending modulus of every group had no difference. In the release test,the elution rate in every group increased progressively with the content of vancomycin in polymethylmethacrylate at first week.When the vancomycin in the content of bone cement attained from 1.5 to 2.0 g after a week,the quantity of the released vancomycin was higher than the others（P<0.05）,and it was gradually in stability.Conclusion Vancomycin can be released from the bone cement utility.1.5-2.0 g vancomycin are optimal doses to be impregnated in 40 g bone cement and they can improve the mechanical property of the bone cement.

Abstract:Objective To investigate the anti-proliferation effect of Ginseng Rh₂（G-Rh₂）on mouse MFC gastric cells and its mechanism.Methods MFC cells (1.0×10⁹•L^-1) were divided into four gourps:control group and G-Rh₂(3,10,30 mg•L^-1) groups.The cell viability was determined by MTT；the cell cycle was detected by flow cytometry；the protein expression of cyclin D₁ and p27^kip1 we observed respectively by qualitative and quantitative analysis.Results Compared with control group,the cell viabilities decreased gradually with the increasing of dose and time in G-Rh₂(3,10,30 mg•L^-1) groups at different time(1,2,4 h)(P<0.05 or P<0.01)， the ratio ofVCM G₀/G₁ cells increased in a dose-dependent manner(P<0.05 or P<0.01),at the same time,the expression level of cell cycle protein cyclin D₁ decreased (P<0.05 or　P<0.01),and the expression level of p27^kip1 increased (P<0.05 or　P<0.01).Conclusion G-Rh₂ can decease cell viability by inducing apoptosis and arresting cell cycle,and the process may associate with p27^kip1 increase and cyclin D₁ decrease.

Abstract:Objective To observe the effects of ilexonin A (IA) on IL-6 and M-CSF following ballon angioplasty in rabbit common carotide artery to provide experimental basis for percutaneous coronary interventions. Methods 30 Japanese rabbits were fed with high cholesterol food for 4 weeks. Then they were divided into three groups randomly. Each group had ten rabbits. ①Control group: the incision was sew directly after right carotide artery of the rabbit was seeked. ②Balloon dilation group:the proximal of the carotide artery was cuted,the ballon was delivered and distended,after it was drawn repeatly,the incision was closed. ③IA therapy group: operation was the same to the balloon dilation group,then IA was administered in vein.All of them were fed with high cholesterol diet for 4 weeks and the blood samples were collected 1 d before the operation and 1 d,1,2,4 weeks after the operation. The serum IL-6 and M-CSF levels were determined with radioimmunoassay.The pathological changes of injuried artery were observed. Results ①The IL-6 level in balloon dilation group was higher than that in IA therapy group after the operation (P<0.05),and came back later to preoperation level. ②Though the M-CSF level in IA therapy group was increased, but it was lower than that in dilation group (P<0.05) and the time of returning to preoperation level was obviously earlier than that in balloon dilation group (P<0.05). ③The pathological results demonstrated that the artery endothelial in control group was smooth and no foam cell in sight.In bolloon dilation group,the endangium was thickened,the vascular smooth muscle cells proliferated,there were many foam cells and the lumen was constrictive.In IA therapy group,the number of foam cells reduced,the constrictive degree of lumen was alleviated.Conclusion IA can redece the levels of serum IL-6 and M-CSF and inhibit the proliferation of vascular smooth muscle cells following ballon angioplasty.

Abstract:Objective To investigate the inhibitory effect of arsenic trioxid（AS₂O₃）on the proliferation of RSC-364 synoviocyte lines stimulated by HMGB1 and its possible mechanism. Methods RSC-364 synoviocytes were cultivated with standard medium as control group or with medium supplemented with HMGB1(HMGB1 group) or HMGB1 and 0, 5,10, 20, 40，80 μmol•L^-1 AS₂O₃(experimental groups),respectively. MTT assay was carried out to study the inhibitory effects of AS₂O₃ with different concentrations on the growth of RSC-364. RT-PCR was used to detect the mRNA expression of STAT1. Proteins of phospho-STAT1, cyclin D1 and proliferation cell nuclear antigen (PCNA) were detected by immunocytochemistry and FCM.Results ① MTT data indicated AS₂O₃ could inhibit the growth of RSC-364 stimulated by HMGB1 in a time-dependent and dose-dependent manner. ②Compared with control group, HMGB1 up-regulated the expression of phospho-STAT1 protein and mRNA as well as PCNA protein. Both mRNA and protein levels of phospho-STAT1 were decreased in the RSC-364 cells exposed to AS₂O₃ in a dose-dependent manner; AS₂O₃ also down-regulated the expression of PCNA protein. ③ HMGB1 up-regulated the expression of cyclin D1, while AS₂O₃ decreased the expression of cyclin D1 .④ There was positive correlation between phospho-STAT1 and cyclin D1 (r=0.842,P=0.001); Conclusion Arsenic trioxide could inhibit the proliferation of RSC-364 synoviocytes, partly by down-regulating the expression of phospho-STAT1/cyclin D1 .

Abstract:Objective To observe the mechanism of action of panax quinquefolium diolsaponins (PQDS) on anti-ventricular remodeling in rats. Methods The model of pressure-loading ventricular remodeling was established through the method of rat’[KG-*3]s abdominal aorta deligation. The male Wistar rats were divided into 5 groups,including sham operation group,remodeling model group,Benazepril group,and low and high dose groups of PQDS. The rats were treated with PQDS (with dose of 50 and 100 mg•kg^-1 i.g) for 6 weeks. The rats in sham operation group and remodeling model group were treated with normal sodium (with dose of[JP3] 10 mL•kg^-1•d^-1 i.g)[JP] for 6 weeks. The rats in masculine medicine group were treated by Benazepril (with dose of 10 mg•kg^-1 i.g) for 6 weeks. After 6 weeks of treatment,myocardial morphological parameters,the content of malondialdehyde (MDA) and activity of superoxide dismutase (SOD) in serum,the contents of prostacycline (PGI₂),thromboxane A₂ (TXA₂),nitric oxide (NO) and angiotensinⅡ (AngⅡ) in plasma were determined. At the same time,the contents of endothelium (ET) in plasma and myocardium were also determined. Results Compared with remodeling model group,the ventricular weight and cardiac coefficient were decreased significantly in the rats treated with PQDS (P<0.05),the content of MDA in serum was declined and the activity of SOD in serum increased signficatly(P<0.05),the contents of AngⅡ and TXA₂ in plasma were declined while the content of PGI₂ in plasma and PGI₂/TXA₂ ratio were increased significatly (P<0.05 or P<0.01). In addition,the contents of ET in plasma and myocardium were also declined markedly (P<0.05 or P<0.01),but the content of NO in plasma had no significant change (P>0.05). Conclusion PQDS has protective effects on ventricular remodeling through increasing anti-oxidase activity,reducing the damage of free radicals and vasoactive substance on myocardium and correcting disequilibrium of PGI₂/TXA₂ in ventricular remodeling etc.

Abstract:Objective To study the protective effects of the polyphenols of vitis amurensis Rupr(PVAR) on rat heart mitochondria injury induced by oxygen radicals.Methods Experiment was designed into 5 groups:normal group,injury group and 25,50,100 mg•L^-1PVAR+injury group.In experiment of rat heart mitochondria injury in vitro,vitamin C and FeSO₄ played as an injury agent,PVAR played as a protectiveagent.The cardiolipin and MDA level of the mitochondria were determined.The membrane fluidity,ATPase activity and swelling of the mitochondria were examined.Results Compared with injury group,the cardiolipin was increased(P<0.05),the MDA was decreased(P<0.05),the membrane fluidity and the ATPase activitiy were increased(P<0.01) in 25,50,100 mg•L^-1 PVAR groups.There was no significant difference of swelling of injured mitochondria between PVAR( 100 mg•L^-1) group and control group.Conclusion Oxygen radicals can result in severe mitochondria injuried.The PVAR can protect the injuried rat heart mitochondria by antagonizing the free radicals.

Abstract:Objective To evaluate the effects of alcohol extract of juglans mandshurica maxim (AEBJ) on immune function of ionizing irradiated mice．Methods The mice were randomly divided into normal control group,irradiating control group,low AEBJ（300 mg•kg^-1）irradiating group,high AEBJ（600 mg• kg^-1）irradiating group,low AEBJ plus drugs only group and high AEBJ plus drugs only group．The number of WBC and LYMPH,LYMPH%,viscera index,ability of lymphocytes transformation were examined．Results Compared with normal control group，the number of WBC and LYMPH,LYMPH%,viscera index,ability of lymphocytes transformation in irradiating control group were decreased significantly (P< 0.05)．Compared with irradiating control group，the number of WBC and LYMPH,LYMPH%,viscera index,ability of lymphocytes transformation in low AEBJ irradiating group and high AEBJ irradiating group were increased significantly (P< 0.05)．Compared with normal control group，LYMPH% and ability of lymphocyte transformation in low AEBJ plus drugs only group and high AEBJ plus drugs only group were increased significantly (P< 0.05)．Conclusion AEBJ　 has protective effects on immune function of ionizing irradiated mice．

Abstract:Objective To investigate the proliferation inhibition of chlorpromazine combined with taxol on Hep-2 cells (human laryngeal carcinoma cell line) and the effects on cell cycle progression. Methods Hep-2 cells at logarithmic growth phase were divided into taxol groups(3.0,6.0 and 12.0 mg•L^-1),chlorpromazine(12.0 mg•L^-1) comined with taxol (4 mg•L^-1)group and control group (100 mL culture fluid).MTT and flow cytometry were used to detect the proliferation inhibition rates of Hep-2 cells in various groups.Flow cytometry was also used to analyze the cell cycle progression of Hep-2 cells and apoptotic rate after administration.Results The proliferation inhibition rates in 3.0，6.0 and 12.0 mg•L^-1 taxol groups were 14.0%,23.9% and 36.7%，respectively,there were significant differences between three groups(P<0.05)；the proliferation inhibition rate in combined group(46.3%) was higher than those in taxol groups(P<0.05).In taxol groups.the number of G₁ phase cells decreased and the number of S and G₂ phase cells increased with the increasing of concentration of taxol.Compared with control group,the apoptotic rates in taxol groups increased(P<0.05). Conclusion Within certain concentration range,chlorpromazine could enhance taxol-induced proliferation inhibition rate,cell cycle progression and apoptotic rate of Hep-2 cells,chlorphomaxine and taxol have significant cooperation effect.

Abstract:Objective To explore the effects of motion environment on memory capacity in low-level lead exposure mice and mechanism.Methods Healthy weaned male ICR mice were randomly divided into control group and experimental group (lead exposure group) and then every group was divided into 2 groups: common feeding group and common feeding plus motion group (n=10). The mice in experimental group were fed with 0.2％ low-level lead drinking water for 8 weeks. Luminosity test (using an atom to absorb a beam split),Morris water maze test and immune test were applied respectively to detect the levels of blood lead and hippocampus lead and the impact of lead on mouse space memory and the histological changes. Results The lead levels in blood and hippocampus in experimental group were significantly higher than those in control group (P<0.01). The lead level in hippocampus in common feeding group of experimental group was higher than that in common feeding plus motion group（P<0.05）. In space memory test,the latent time to reach refuge platform in common feeding group of experimental group was slower than that in control group (P<0.05),and it was higher in common feeding group of experimental group than that in common feeding plus motion group(P<0.05). The pathohistological results showed that focal necrosis was found in common feeding groups of experimental group,and focal uneven distribution,degeneration of never cells were found in common feeding plus motion group of experimental group . Conclusion The stimulation of motion environment can improve the ability of space memory and promote the hippocampus plasticity and compensatory in low-level lead exposure mice.

Abstract:Objective To observe the effects of estrogen on behavior and 5-HT in periaqueductal gray （PAG）in migraine model rats. Methods Tewnty-four ovariectomized Wistar rats were divided randomly into four groups：control group (Group A)，migraine group(Group B)，low dose estradiol-treated ovariectomized group(Group C)， high dose etradiol-treated ovariectomized group(Group D).After 1 week，the rats in Group B,C and D were injected with nitroglycerine 10 mg•kg^-1 ubcutaneously to make migraine rat models, the rats in Group A were given peanut oil alike，and the behavior changes were observed.2 h after injection,the rats were killed and the midbrains were separated and then 5-HT immunohistochemical staining was performed.Results Behavior: compared with Group B，the degrees of red-calws,red-ears and red-tail rats in Group D relieved obviously，the times of climbing hutch and scratching head were much fewer, while the rats in group C showed no significant difference;Immunohistochemical staining:compared with Group A，the 5-HT-positive neurons expression in PAG of Group B and C were more obviously（P<0.01）, especially in the ventrolateral and dorsolateral of PAG,the degree of 5-HT-positive neurons increased in PAG of Group B as most,Group C as more,while group D as a little. There were significant differences between various groups(P<0.01). Conclusion Estrogen can reduce the degree of 5-HT-positive neurons activating in PAG,and its role in a dose-dependent manner,and high dose estrogen can decrease the changes of behavior of migraine rats, the mechanism may be related with that estrogen can influence the neuroactive substances taking part in migraine.

Abstract:Objective To investigate the expression of Survivin in primary hepatocellular carcinoma（HCC）and its relationship with clinical pathological features and explore the role of Survivin in the development of HCC. Methods The expressions of Survivin were detected in 30 cases of HCC,20 cases of para-cancer tissue and 10 cases of normal liver tissue by flow cytometry (FCM) and immunohistochemistry (SP). The clinical data were analyzed retrospectively. Results ① The expression of Survivin in the HCC tissue was significantly higher than those in para-cancer tissue and normal liver tissue (P<0.05);②The expression of Survivin was negative in normal tissue surrounding cancer; ③The expression of Survivin was independence of tumor size,location,capsule,AFP (P> 0.05),while the expression of Survivin was related with endoscopic portal vein thrombosis formation (P<0.05). Conclusion The hyperexpression of Survivin in tissue can reflex the biological behavior of HCC. The specificness of survivin protein expression make it to be new target of tumor diagnosis and treatment.

Abstract:Objective To study the expression and regulation characteristics of aquaporin water channel(AQP) of cerebuller metastatic tumor and brain tissue surrounding tumor and to understand the role of abnormal expression of AQP in formation and elimination of brain edema.Methods The tumor tissue specimens from five lung adencarcinoma brain metastasis cases after surgical resection were accessed.The total RNA was extracted,and RT-PCR，immunohistochemical staining were progressed to study the expression and regulation characteristics of AQP.The average gray values of near and far regions from tumor tissue were measured with HPIAS-1000 cells measuring procedure to compare actual expression quantity of AQP.Results AQP4 had a high expression in the peritumoral brain tissue and no expression in the center of brain metastasis tumor organization.The AQP4 staining was junior in the more distant region from tumor and it added significantly in the region close to the tumor tissue.Stained AQP1 was not found in cerebullar metastatic tumor and peritumoral brain microvascular endothelial cells.Conclusion The expression of AQP4 is increased significantly in the region next to the cerebullar metastatic tumor tightly.It is probably related to the formation of peritumoral brain edema.The negative expression of AQP1 might not be conducive to the removal of edema in the interspace of nerve cells.

Abstract:Objective To evaluate the hemodynamic responses of esmolol to nasotracheal intubation with fiberbronchoscope(FOB). Methods Thirty-five ASAⅠorⅡpatients undergone stomatology and otorhinolaryngology surgery were randomly divided into fiberoptic nasotracheal intubation esmolol group (esmolol group) and fiberoptic nasotracheal intubation group (control group). The intravenous administration of esmolol 1mg•kg^-1 was performed 2 min before tracheal intubation in esmolol group. Noninvasive SBP,DBP,MBP,HR and SpO₂ were recorded before and after anesthetic induction,at intubation and 1,2,3,4,5 min after intubation. Results The SBP,DBP and MBP 1 min after intubation in esmolol group were significantly lower than those in control group(P<0.05). The HR at intubation in esmolol group was significantly lower than that in control group(P<0.05). The maximums of SBP,DBP,MBP and HR in esmolol group were also lower than those in control group(P<0.05). The SpO₂ levels in two groups were above 96% during the test. Conclusion General anesthesia of clinical standard depth cannot inhibit effectively the increase of blood pressure and heart rate caused by nasotracheal intubation through FOB;Introvenous administration of esmolol 1 mg•kg^-1 2 min before tracheal intubation can effectively inhibit cardiovascular responses caused by nasotracheal intubation through FOB.

Abstract:Objective To develop a new system of computer-aided design(CAD) for individual metacarpophalangeal joint prosthesis based on rapid prototyping technique. Methods A hand of cadaver was scanned with PLUS-4 spiral computed tomography (CT).Then the transaxial 2D image data of digitus metacarpophalangeal joint were reconstructed into 3D digitized contour data by 3DMSR that was designed by ourselves. Then,the intramedullary stem was designed in software of Surface 9.0. Results The 3D contour image of metacarpophalangeal joint presented was reconstructed by 3DMSR and edited by Surface 9.0 easily for CAD of individual metacarpophalangeal joint.The intramedullary cavity was like choanoid. The intramedullary stem longitude of articular head and fossa were 47.31 and 35.20 mm.The intramedullary stem fit cavity.The model fit anatomical shape. Conclusion The 3D contour image of metacarpophalangeal joint can be obtained by spiral CT scanning，and the digitized data can be applied directly to CAD of individual artificial joint and subsequently rapid prototyping fabricating．In addition，the reconstruction method is simple and can be applied widely to clinical implant fabricating practice of orthopaedics．

Abstract:Objective To investigate the effects of different surgical treatments on deep venous thrombosis(DVT) in the lower extremities of the old patients who underwent femoral neck fracture in order to provide basis for prevention of DVT with selective anticoagulant. Methods 180 old patients with new femoral neck fracture without any drug-anticoagulation therapy were divided into three groups according to different operative model. Group A: 40 cases were operated by closed reduction and internal fixation with hollow screws;group B: 68 cases were operated by replacement of total hip;group C: 72 cases were done by artificial femoral head replacement. Ultrasonic Doppler test was performed when swelling and/or pain,with or without Homans sign/Neuhofs sign were found positive in the operative legs. Results Doppler showed that 10 cases were positive in group A,the incidence of DVT was 25.0%;in group B 29 cases were positive,one case who was negative died for mixed DVT complicating pulmonary embolism in injuried extremity confirmed by autopsy prior to discharge,the incidence of DVT was 44.1%;in group C 20 cases were positive,the incidence of DVT was 27.8%. The incidence of of DVT in group A and group C were lower than that in group B( P<0.05),there was no significant difference between group A and group C.Conclusion The replacement of total hip may lead to a higher incidence of DVT,but the closed reduction and internal fixation with hollow screws and artificial femoral head replacement make a lower one in the old patients with femoral neck fracture.

To investigate the psychological factors related to internet addiction disorder(IAD) in Chinese college students and compare the differences in sensation seeking and biological traits and basis between IAD and common students. Methods 1 784 college students were chosen randomly and interviewed with Sensation Seeking Scale and Questionnaire on IAD. Then ten IAD and ten no IAD were undergone long latency auditory evoked potential studies using tones at 90 dB SPL. Results There was significant difference between males and females（P<0.05）,but none among grades. There were significant differences in sensation seeking total scores and Dis scores（P<0.05）.N1 amplitude at Fz of IAD was more higher than that of no IAD（P<0.05）.There was relationship between IAD and sensation seeking（r＝0.340，P<0.01）. Conclusion There are differences between IAD and common students in sensation seeking and biological traits and basis.The sensation seeker’〖KG-*3〗s mental life should be paid more attention to avoid IAD.