2012年02期目次

  • Expression of human papillomavirus 18L1 protein in Pichia pastoris and immunogenicity of its virus-like particles

    SHEN Qiong,LEI Jian-qiang,ZHOU Chao-ming,ZHANG Gao-xia(Shanghai Zerun Biotech Co.,Ltd.,Shanghai 201203,China)

    Objective To express human papillomavirus(HPV) 18 L1 protein in Pichia pastoris,purify the virus-like particles(VLPs) and determine its immunogenicity.Methods Recombinant plasmid pHPV18 L1 was transformed to P.pastoris X-33 by electrotransformation and induced with methanol.The expressed recombinant HPV18 L1 protein was identified by SDS-PAGE and Western blot,then purified by ion exchange chromatography and molecular sieve chromatography,and analyzed for purity by SEC-HPLC,for particle size distribution and structure by dynamic light scattering and transmission electron microscopy,and for immunogenicity by determination of ED50 in mice and antibody titer in rats.Results HPV18 L1 protein,with a relative molecular mass of about 55 000,was effectively expressed in P.pastoris,which showed specific binding to mouse anti-HPV18 L1 monoclonal antibody.The purified HPV18 L1 protein reached a purity of 99%,of which the VLPs were even in size,at a diameter of about 50 nm.The HPV18 L1 protein after adsorption with aluminum hydroxide induced high antibody titer in rats,of which the ED50 in mice was 0.006 68 μg.Conclusion Self-assembled HPV18 L1 was highly expressed in P.pastoris,of which the VLPs showed high immunogenicity after purification and adsorption with aluminum hydroxide.It laid a foundation of industrial production of HPV18 L1 vaccine.

    2012 02 v.25 [Abstract][OnlineView][Download 364K]

  • Preparation of inactivated Japanese encephalitis vaccine by culture of Vero cells in WAVE bioreactor

    TONG Fang,ZHAO Yu-xiu,WANG Hui,LIANG Hong-yang,MA Ke,MA Le,NIU Zhi-bin,YANG Yang,LIU Ying,ZHANG Yue-lan,(Beijing Tiantan Biological Products Co.Ltd.,Beijing 100024,China)

    Objective To prepare inactivated Japanese encephalitis(JE) vaccine by culture of Vero cells in WAVE bioreactor.Methods Vero cells were cultured on microcarriers in 2 L WAVE bioreactor by semi-continuous culture,from which samples were taken every 24 h and observed for growth,and monitored for glucose concentration until the cell monolayer was formed.JE virus was inoculated onto the cell monolayer at a MOI of 0.03 ～ 0.5,and harvested when the CPE rate reached 25%,which was purified to prepare inactivated JE vaccine.Overall control tests were performed on the prepared vaccine according to the requirements in Chinese Pharmacopeia(Volume Ⅲ,2010 edition).Results Vero cells formed a dense monolayer on microcarriers 6 d after culture,of which the CPE rate reached 25% 2 d after inoculation with JE virus while the virus bulk was harvested at a rate of 1 L/d for 5 ～ 7 d,with a total volume of 5 ～ 7 L.The virus titer reached a peak value of 8.54 LgLD50/ml on day 4 after culture.All the quality indexes of prepared vaccine met the requirements in Chinese Pharmacopeia(VolumeⅢ,2010 edition).Conclusion A method for preparation of inactivated JE vaccine by culture of Vero cells in WAVE bioreactor was preliminarily developed.

    2012 02 v.25 [Abstract][OnlineView][Download 293K]

  • Construction and in vitro expression of plasmid pVAX1-Rv3407 as a DNA vaccine against tuberculosis

    LIU Rong-na*,FANG Xi-jing,ZHANG Ai-hua,YANG Xiao-ming,ZHANG Ya-ting,BI Lan(*Wuhan Institute of Biological Products Co.,Ltd.,Wuhan 430060,China)

    Objective To construct plasmid pVAX1-Rv3407 as a DNA vaccine against tuberculosis and determine its expression in mammal cells 293T.Methods Rv3407 gene was amplified by PCR using the AERAS-422 genome of recombinant BCG(rBCG) as a template,based on which recombinant plasmid pVAX1-Rv3407 was constructed and transfected to 293T cells.The expression of Rv3407 protein was identified by IFA and Western blot.Results Rv3407 gene fragment at a length of 300 bp was amplified by PCR.Restriction analysis and DNA sequencing proved that recombinant plasmid pVAX1-Rv3407 was constructed correctly.Rv3407 protein was effectively expressed in 293T cells 48 h after transfection,which was mainly distributed in cytoplasm.Conclusion Recombinant plasmid pVAX1-Rv3407 as a DNA vaccine against tuberculosis was successfully constructed,which laid a foundation of developing novel tuberculosis vaccine.

    2012 02 v.25 [Abstract][OnlineView][Download 217K]

  • Construction and identification of lentiviral expression vector hsa-miR-150

    ZHANG Jun*,ZHANG Tao,WANG Bo,LI Shao-lin(*Department of Radiology,Chongqing Medical University,Chongqing 400016,China)

    Objective To construct lentiviral expression vector hsa-miR-150 and determine the expression of miR-150 in infected hepatocellular carcinoma cell line Hep3B.Methods The hsa-miR-150 gene fragment was synthesized chemically and cloned into lentiviral vector pGIPZ.The constructed recombinant plasmid pGIPZ-hsa-miR-150 was transfected to packaging cell line 293T,and the packaged virus was determined for titer.Recombinant lentivirus particles were harvested and concentrated,then stably transfected to Hep3B cells and determined for expression rate of GFP by flow cytometry,and for expression level of miR-150 by fluorescent quantitative PCR.Results PCR amplification of bacterial colony and sequencing proved that recombinant lentiviral expression vector pGIPZ-hsa-miR-150 was constructed correctly.The titer of recombinant lentivirus was 5.7 × 108/ml.The expression rate of GFP in Hep3B cells stably transfected with pGIPZ-hsa-miR-150 was(97.78 ± 1.14)%,while the expression level of miR-150 increased by 6-folds as compared with those in blank control group(P < 0.05).Conclusion The lentiviral expression vector hsa-miR-150 was constructed successfully and highly expressed in Hep3B,which laid a foundation of further study on biological function of miR-150.

    2012 02 v.25 [Abstract][OnlineView][Download 293K]

  • Prokaryotic expression and purification of human S-adenosylmethionine decarboxylase

    ZHANG Jun,HAN Yu,CAI Fu-qiang,QIN Ye,XIA Yan,WANG Yan-lin(Medical College,Institute of Molecular Biology,China Three Gorges University,Yichang 443002,Hubei Province,China)

    Objective To clone the cDNA of human S-adenosylmethionine decarboxylase(AdoMetDC),express in prokaryotic cells and purify the expressed protein.Methods Human AdoMetDC cDNA was cloned from the total RNA of HeLa cells by nested RT-PCR and inserted into vector pET-15b.The constructed recombinant plasmid pET-15b/AdoMetDC was transformed to competent E.coli Rossetta(DE3) and induced with IPTG.The expressed recombinant protein was purified by Ni-NTA chromatography at condition of denaturation with urea,and identified by SDS-PAGE and Western blot.Results The full-length cDNA sequence of human AdoMetDC was cloned,and recombinant plasmid pET-15b/AdoMetDC was constructed.The expressed recombinant AdoMetDC protein showed bands of proenzyme as well as subunits α and β obtained by autocatalytic cleavage,with relative molecular masses of about 35 000,30 000 and 14 000 respectively,on SDS-PAGE profile.Proenzyme and subunit β were effectively purified by Ni-NTA chromatography.The purified recombinant protein was specifically recognized by the antibodies against His and AdoMetDC.Conclusion Human AdoMetDC was expressed in prokaryotic cells and purified,which laid a foundation of study on its function and clinical application.

    2012 02 v.25 [Abstract][OnlineView][Download 248K]

  • Construction of siRNA expression vector for JTV1 gene and its effect on proliferation of K562 cells

    WU Yan*,LIU Bei-zhong,WANG Chong,ZHONG Liang,ZHU Dan,WANG Chun-guang,LI Liang,GAO Yan-jun(*Central Laboratory of Yongchuan Hospital Affiliated to Chongqing Medical University,Chongqing 400016,China)

    Objective To construct the siRNA expression vector for JTV1 gene,transfect to K562 cells and identify its interference effect.Methods The siRNA interfering sequence targeting to JTV1 gene was synthesized and cloned into vector pGeneSil-1.The constructed recombinant plasmid pGeneSil-1-JTV1-1.1 siRNA was transfected to human K562 cells.The positive clones were screened with G418,based on which the effect of recombinant plasmid pGeneSil-1-JTV1-1.1 siRNA on transcription and translation of JTV1 gene in K562 cells was investigated by RT-PCR and Western blot,and that of JTV1 gene expression on proliferation of K562 cells by MTT method.Results Sequencing proved that recombinant plasmid pGeneSil-1-JTV1-1.1 siRNA was constructed correctly.Both the transcription and translation levels of JTV1 gene in K562 cells transfected with the recombinant plasmid decreased significantly.The inhibition of JTV1 expression promoted the proliferation of K562 cells.Conclusion Recombinant plasmid pGeneSil-1-JTV1-1.1 siRNA was successfully constructed,and stable K562 cell clones in which the expression of JTV1 gene was inhibited were obtained,which laid a foundation of further study on function of JTV1 gene and its correlation to tumor.

    2012 02 v.25 [Abstract][OnlineView][Download 484K]

  • Prokaryotic expression and identification of VP3 gene of group A human rotavirus

    ZHANG Shun,PAN Xiao-xia,YUAN Jing,WEN Yu-ling,CHEN Yuan-ding(Institute of Medical Biology,Chinese Academy of Medical Sciences & Peking Union Medical College,Kunming 650118,China)

    Objective To clone the gene encoding structural protein VP3 of group A human rotavirus(RV) TB-Chen strain,express in prokaryotic cells and investigate its molecular phylogenesis and genotype.Methods The gene encoding VP3 of TB-Chen strain was amplified by PCR and cloned into pETL vactor.The constructed recombinant plasmid pET-VP3 was transformed to E.coli BL21(DE3).The expressed recombinant protein was identified by SDS-PAGE and Western blot,based on which the cloned VP3 gene was analyzed for molecular phylogenesis and genotype.Results Restriction analysis and sequencing proved that recombinant plasmid pET-VP3 was constructed correctly.The expressed recombinant protein,with a relative molecular mass of about 98 000,existed in a form of inclusion body and was recognized by the guinea pig sera against whole virus of SA11 strain.By the so far,the discovered RV VP3 was of seven genotypes,of which those from TB-Chen and SA11 strains were belonged to genotypes M2 and M5 respectively.Conclusion The VP3 protein of group A human RV TB-Chen strain was successfully expressed in prokaryotic cells,which laid a foundation of further study on structure and function as well as development of VP3.

    2012 02 v.25 [Abstract][OnlineView][Download 409K]

  • Construction of fluorescent eukaryotic expression vector for hepatitis B virus x protein and its effect on human normal liver cell strain L02

    QIAN Guan-hua*,DONG Jun-jun,DUAN Chang-zhu,PENG Hui-min(*Department of Cell Biology and Genetics,Chongqing Medical University,Chongqing 400016,China)

    Objective To construct a fluorescent eukaryotic expression vector for hepatitis B virus(HBV) x protein and determine its effect on proliferation of human normal liver cell strain L02.Methods Full-length sequence at encoding region of HBx gene was amplified by PCR using plasmid pcDNA3-HBV as a template,and cloned into fluorescent expression vector pIRES2-EGFP.The constructed recombinant plasmid pIRES2-EGFP-HBx was tranfected to L02 cells,based on which the cells stably expressing HBx were screened,and determined for transcription level of HBx mRNA by RT-PCR,for expression level of HBx protein by Western blot,and for proliferation activity by MTT method.Results Restriction analysis and sequencing proved that recombinant fluorescent eukaryotic expression vector pIRES2-EGFP-HBx was constructed correctly.Both the transcription of HBx mRNA and expression of HBx protein were proved in the L02 cells transfected with plasmid pIRES2-EGFP-HBx.The proliferation activity of L02 cells transfected with plasmid pIRES2-EGFP-HBx was significantly higher than that with empty vector pIRES2-EGFP.Conclusion The fluorescent eukaryotic expression vector for HBV x protein was successfully constructed,and L02 cell strain stably expressing HBx was screened,which laid a foundation of further study on effect of HBx on regulatory pathway of cell cycle as well as the molecular mechanism of HBV-associated HCC caused by HBx.

    2012 02 v.25 [Abstract][OnlineView][Download 308K]

  • Expression of activin receptor-interacting proteins 1,2 in mouse macrophages

    CUI Xue-ling*,GE Jing-yan,LI Chen-guang,SUN Yang,NIU Li-man,LIU Hai-yan,LIU Zhong-hui,WANG Yi-nan(*Department of Genetics,Norman Bethune College of Medicine,Jilin University,Changchun 130021,China)

    Objective To investigate the expression and role of activin receptor-interacting proteins 1,2(ARIP1,2) in mouse macrophages.Methods The expression of ARIP1,2 was determined by immunocytochemical staining.RAW264.7 cells were co-transfected with plasmids CAGA-lux and CMV-gal,plus plasmids pcDNA3-ARIP1,pcDNA3-ARIP2 or empty plasmid pcDNA3,respectively,then stimulated with activin A,and determined for transcription activity of report gene.The expression of ActRⅡA mRNA was determined by RT-PCR.Results Immunocytochemical staining proved that ARIP1,2 were expressed in RAW264.7 cells.The overexpressions of ARIP1,2 inhibited the transcription of specific gene induced by activin A.The overexpression of ARIP2 inhibited,while that of ARIP1 showed no significant effect on the expression of ActRⅡA mRNA in RAW264.7 cells.Conclusion ARIP1,2 were coexpressed in mouse macrophages,which down-regulated the signal transduction of activin by different action modes.

    2012 02 v.25 [Abstract][OnlineView][Download 297K]

  • Establishment of lung cancer cell strain for stable expression of RAS guanine nucleotide releasing factor 2

    WANG Yong*,CHEN Hong,BU You-quan(*Department of Respiratory Medicine,The First Affiliated Hospital of Chongqing Medical University,Chongqing 400016,China)

    Objective To construct a eukaryotic expression vector for RAS guanine nucleotide releasing factor 2(RASGR-F2),tagged with GFP,and establish a lung cell strain for stable expression of RASGRF2.Methods Plasmid RASGRF2-pCMV6-Myc-DDK was digested with SgfⅠand NotⅠ,and RASGRF2 gene fragment was recovered and subcloned into vector pCMV6-GFP.Lung cancer H1299 cells were transfected with the constructed recombinant plasmid RASGRF2-pCMV6-GFP and observed for location of RASGRF2 protein under invert fluorescent microscope.The cell strain for stable expression of RASGRF2 was established by G418 screening,and the expression of RASGRF2 was determined by RT-PCR and Western blot.Results Restriction analysis and sequencing proved that RASGRF2 gene was successfully cloned into eukaryotic expression vector pCMV6-GFP.Recombinant RASGRF2-GFP protein was mainly expressed in cytoplasm of H1299 cells.RT-PCR and Western blot showed that RASGRF2 was stably expressed in H1299 cells.Conclusion The lung cancer cell strain for stable expression of RASGRF2 gene was successfully established,which laid a foundation of further study on function of RASGRF2 gene.

    2012 02 v.25 [Abstract][OnlineView][Download 267K]

  • Cloning and prokaryotic expression of full-length gene of human granzyme A

    DU Jia-ni,CHEN Lei,LONG Feng-ying,JIANG Wen-zheng(School of Life Science,East China Normal University,Shanghai 200062,China)

    Objective To clone human granzyme A(GzmA) gene,express in E.coli and preliminarily optimize the condition for expression.Methods Human GzmA gene was amplified by RT-PCR and inserted into prokaryotic expression vector pET24a(+).The constructed recombinant plasmid pET24a-GzmA was transformed to E.coli BL21(DE3) and induced with IPTG.The expressed product was identified by SDS-PAGE and Western blot.The temperature,IPTG concentration and time for induction as well as the A600 value of bacterial liquid when the induction was started were optimized.Results Restriction analysis and sequencing proved that GzmA gene with correct sequence was inserted into vector pET24a(+).The expressed recombinant protein,with a relative molecular mass of about 29 000,showed specific binding to mouse anti-His monoclonal antibody.The optimal temperature for induction of recombinant E.coli was 37℃.However,IPTG concentration,time for induction and the A600 value of bacterial liquid when the induction was started showed little effect on expression level of recombinant protein.Conclusion Human GzmA gene was successfully cloned and expressed in E.coli.

    2012 02 v.25 [Abstract][OnlineView][Download 248K]

  • Role of Hedgehog signaling pathway in invasion and metastasis of gastric cancer and relevant molecular mechanism

    HAO Ya-qin*,OUYANG Xiao-bo,DAI Jian-bo,WANG Li(*Department of Gastroenterology,The First Affiliated Hospital of Chongqing Medical University,Chongqing 400016,China)

    Objective To investigate the roles of Hedgehog(Hh) signaling pathway and epithelial-mesenchymal transition(EMT) in invasion and metastasis of gastric cancer as well as the relevant molecular mechanism.Methods Human gastric carcinoma SGC-7901 cells were treated with cyclopamine,Snail siRNA,empty plasmid and cyclopamine + Snail siRNA for 48 h respectively,using those in routine culture as control.The cells in various groups were determined for transcription levels of Gli1,Snail and E-cadherin mRNAs by RT-PCR,for invasion ability by Transwell test,and for effect on adhesion ability to foreign matter by adhesion test on Marigel glue.Results Compared with those in control group,the transcription level of Gli1 mRNA in cyclopamine and cyclopamine + Snail siRNA groups decreased significantly(P < 0.05);however,the transcription levels of Snail mRNA in cyclopamine,Snail siRNA and cyclopamine + Snail siRNA groups decreased significantly,while that of E-cadherin mRNA increased significantly(P < 0.05).Both the invasion abilities and adhesion abilities to foreign matters of cells in cyclopamine,Snail siRNA and cyclopamine + Snail siRNA groups decreased significantly as compared with those in control group(P < 0.05).Conclusion Hh signaling pathway may be involved in regulation of Snail by Gli1 to low the expression of E-cadherin,which results in EMT,thus plays an important role in the invasion and metastasis of gastric cancer.

    2012 02 v.25 [Abstract][OnlineView][Download 411K]

  • Effect of bone marrow mesenchymal stem cells on experimental liver fibrosis in rats and relevant mechanism

    ZHOU Wei*,CHEN Peng-fei,WU Xiao-ling,JIANG Rong,XU Yan-hua(*Department of Gastroenterology,The Second Hospital Affiliated of Chongqing Medical University,Chongqing 400010,China)

    Objective To investigate the effect of bone marrow mesenchymal stem cells(BMSCs) transplanted on experimental liver fibrosis in rats as well as the relevant mechanism.Methods BMSCs were isolated and purified from male SD rats by direct adherence method.Thirty female SD rats were divided into normal control,liver model and BMSC groups.The rats in model and BMSC groups were injected s.c.with 40% carbon tetrachloride,3 times a week,to copy liver fibrosis model.Eight weeks after starting copy of the model,the rats in BMSC group were transplanted twice with 2 × 106 BMSCs,at an interval of 3 d.The rats in various groups were killed 12 after starting copy of the model,of which the alanine amiotransferase(ALT),asparatate aminotransferase(AST) and albumin(ALB) content in sera were determined,the pathological change of liver was observed by HE and VG staining,the expressions of typeⅠ collagen(ColⅠ) and glial fibrillary acidic protein(GFAP) in liver were determined by immunohistochemical assay and fluorescent quantitative PCR,and those of transformation growth factor(TGF)β1 and Smad3 mRNAs by fluorescent quantitative PCR.Results The liver fibrosis of rats was severer in BMSC group than in model group.Compared with those in model group,the ALT and AST levels in BMSC group increased significantly(P < 0.05),while ALB level decreased significantly(P < 0.05).Co1Ⅰ and GFAP were expressed in a large quantity in fibers of rats in BMSC group,of which the mRNA and protein levels were significantly higher in BMSC group than in model group(P < 0.05).The expression levels of TGFβ1 and Smad3 mRNAs were significantly higher in model and BMSC groups than in normal group(P < 0.05),and in BMSC group than in model group(P < 0.05).Conclusion The transplantation with BMSCs aggravated the liver fibrosis of rats by up-regulating the expressions of TGFβ1 and Smad3 in TGFβ/Smad signal transduction pathway.

    2012 02 v.25 [Abstract][OnlineView][Download 360K]

  • Expressions of interleukin-17,transforming growth factor-β and interleukin-6 mRNAs in mouse spleen infected with Mycoplasma suis

    TIAN Jing*,TANG Xin,BA Cai-feng,YANG Lei-fang(*Experimental Animal Center,Liaoning Medical College,Jinzhou 121001,Liaoning Province,China)

    Objective To determine the changes of interleukin-17(IL-17),transforming growth factor-β(TGF-β) and interleukin-6(IL-6) mRNAs in spleens of BALB/c mice infected with Mycoplasma suis.Methods BALB/c mice were infected with purified M.suis,using physiological saline as control.The spleens of mice were collected aseptically on days 1,3,5,7 and 9 after infection respectively,and determined for expressions of IL-17,TGF-β and IL-6 mRNAs by RT-PCR.Results The expression levels of IL-17 and IL-6 mRNAs in spleens of mice on days 1,3,5,7 and 9 after infection were significantly higher than those in control group(P < 0.05),and reached peak values on days 5 and 7 respectively.However,the expression levels of TGF-β mRNA on days 1,3,5 and 7 after infection were significantly higher than those in control group(P < 0.01),and reached the peak value on day 3.The change tendency of IL-17 was positively related(r = 0.568),while that of TGF-β was negatively related to that of IL-6(r =-0.645).Conclusion The IL-17,TGF-β and IL-6 levels increased in the mice infected with M.suis,and reached peak values on days 5,3 and 7 after infection,which provided an experimental basis for mechanism of immunological regulation of M.suis associated diseases and laid a theoretical foundation of clinical immunotherapy.

    2012 02 v.25 [Abstract][OnlineView][Download 311K]

  • Role of resistin in insulin-resistance of rats with nonalcoholic fatty liver disease

    ZHU Liang-rong,GUAN Xiao-qin,QI Ming-mei,WANG Li-juan(Department of Pathology,Chongqing Medical University,Chongqing 400016,China)

    Objective To investigate the role of resistin in insulin-resistance(IR) of rats with nonalcoholic fatty liver disease(NAFLD).Methods Rat model of NAFLD caused by IR was established by feeding with modified high fat diet,using those with basal forage as control.The serum and liver tissue samples were collected 6,8 and 10 weeks after establishment of model and determined for triglyeride(TG),total cholesterol(TC),alanine amiontrasnferase(ALT),aspartata aminotransferase(AST) fasting plasma glucose(FPG) and fasting insulin(FINS) contents in sera,based on which the insulin sensitivity index(ISI) was calculated.The pathological change of liver was observed by HE staining.The mRNA transcription and protein expression levels of insulin receptor substrate-2(IRS-2),resistin and NF-κB in liver tissue were determined by RT-PCR and Western blot respectively,of which the relationship was analyzed.Results The change of general status of rats indicated that the NAFLD model was successfully established.The determination results of TG,TC,ALT and AST and pathological examination of liver proved hyperlipemia,liver function lesion and IR in model rats.Both the mRNA transcription and protein expression levels of either resistin or NF-κB in model group increased significantly as compared with those in control group(P < 0.05),in a time-dependent mode.However,both the mRNA transcription and protein expression levels of IRS-2 decreased gradually as compared with those in control(P < 0.05),in a time-dependent mode.The expression of resistin was positively related to that of NF-κB,while was negatively related to that of IRS-2.Conclusion The onset of IR in NAFLD might be associated with insulin signaling pathway,involving the decrease of IRS-2 and increase of resistin.Resistin not only influenced the normal signal transduction of insulin upstream pathway by inhibiting the phosphorylation of IRS,but also increased the severity of IR by activating NF-κB to inhibit the signal transduction of insulin downstream pathway.

    2012 02 v.25 [Abstract][OnlineView][Download 334K]

  • Construction of eukaryotic expression vector for p16 gene and its expression in osteosarcoma cells

    XIA Yi-fan*,YU Xian,ZHANG Jian(*Department of Orthopaedics,The First Affiliated Hospital of Chongqing Medical University,Chongqing 400016,China)

    Objective To construct a eukaryotic expression vector for p16 gene and determine its expression in osteosarcoma U-2OS cells.Methods Total RNA of HeLa cells was extracted,with which p16 gene was amplified and cloned into eukaryotic expression vector pcDNA3-HA.The constructed recombinant plasmid pcDNA3-HA-p16 was transfected to U-2OS cells,and the expression of p16/HA was identified by IFA and Western blot.Results Restriction analysis and sequencing proved that recombinant plasmid pcDNA3-HA-p16 was constructed correctly.The p16/HA was successfully expressed in U-2OS cells,which was mainly distributed in nucleus.Conclusion The eukaryotic expression vector for p16 gene was successfully constructed and expressed in U-2OS cells.

    2012 02 v.25 [Abstract][OnlineView][Download 259K]

  • Effect of berberine on lipid metabolism and atherosclerosis of atherosclerotic rabbits

    LUO Ying,HE Qi,JIN Lei,ZHOU Qi-xin,YANG Jun-xia(Depatment of Pharmacology,Chongqing Key Laboratory of Biochemistry and Molecular Pharmacology,Chongqing Medical University,Chongqing 400016,China)

    Objective To investigate the effect of berberine on lipids metabolism and atherosclerosis of atherosclerotic rabbits as well as the potential mechanism.Methods Rabbit model of atherosclerosis was established by feeding with high fat diet.The serum total cholesterol(TC),triglycerides(TG),low density lipoprotein-cholesterol(LDL-C) and high density lipoprotein-cholesterol(HDL-C) levels in sera of model rabbits before and after administration with berberine by gavage were determined.The aortic arteries were observed for pathomorphology by HE staining,and determined for inducible nitric oxide synthase(iNOS) activity by iNOS detection kit.The peroxisome proliferator activated receptor γ(PPARγ) mRNA level in adipose tissue was determined by real time fluorescent quantitative PCR method.Results As compared with those of normal rabbits as control,the serum TC,TG and LDL-C levels of model rabbits increased significantly(P < 0.05),while the endangium was thickened,the lipid deposition was observed,the foam cells were formed,and both the iNOS activity in aortic arteries and PPARγ mRNA level in adipose tissue increased significantly(P < 0.05).However,after invention with berberine especially that at a low dosage,the serum TC,TG and LDL-C levels as well as the iNOS activity in aortic arteries and PPARγ mRNA level in adipose tissue decreased significantly(P < 0.05),while the lipid deposition and foam cell formation were relieved.Conclusion Berberine obviously reduced the blood lipid levels and aortic atherosclerosis,of which the mechanisms was associated with the inhibition of iNOS activity and down-regulation of PPARγ mRNA level.

    2012 02 v.25 [Abstract][OnlineView][Download 380K]

  • Inhibition of proliferation and induction of programmed death of Hep-2 cells by FTY720

    ZHANG Shu-fang*,SUN Ji-feng,Ma Ying,TAI Gui-xiang(*Department of Biochemistry,Basic Medical School,Changchun Medicial College,Changchun 130031,China)

    Objective To investigate the inhibition of proliferation and induction of programmed death of Hep-2 cells cultured in vitro by FTY720.Methods Hep-2 cells were treated with FTY720 at various concentrations(800,1 600 and 3 200 ng/ml) for 48 h,then observed for proliferation activity by MTT assay,for morphology by Switzerland-Giemsa staining,and for cell cycle and apoptosis by flow cytometry.Results FTY720 showed dose-dependent inhibitory effect on proliferation of Hep-2 cells.The inhibiting rate of cell proliferation by FTY720 at a concentration of 3 200 ng/ml was(60.900 ± 0.071)%(P < 0.05).Programmed death was observed in the cells treated with FTY720 which arrested the cells at G2 phase.The apoptosis rate of cells treated with 1 600 ng/ml FTY720 increased significantly(P < 0.05).Conclusion The FTY720 at a certain concentration inhibited the proliferation,regulated the cell cycle and induced the programmed death of Hep-2 cells cultured in vitro.

    2012 02 v.25 [Abstract][OnlineView][Download 326K]

  • Effect of tetramethylpyrazine on expression of vascular cell adhension molecule-1 in mice with ulcerative colitis

    CHEN Wen-min,JIANG Qiong(College of TCM,Chongqing Medical University,Chongqing 400016,China)

    Objective To investigate the effect of tetramethylpyrazine(TMP) on expression of vascular cell adhension molecule-1(VCAM-1) in mice with ulcerative colitis(UC) as well as role of TMP in treatment of UC.Methods Mice were randomly divided into normal control,model and TMP groups.Mouse model of UC was established by induction with dextran sulfate sodium(DSS),then observed for inflammation evaluation indexes,such as disease activity index(DAI) as well as morphology and lesion of colon tissue,and determined for expression of VCAM-1 in colon mucosa by immunohistochemical assay.Results The inflammation evaluation indexes of mice in model group were significantly higher than those in normal control group(P < 0.01).Though little expression of VCAM-1 was observed in colon mucosa of mice in normal control group,the expression level increased significantly in model group(P < 0.01) and decreased significantly in TMP group(P < 0.01).Conclusion The expression of VCAM-1 increased in mouse model of UC induced by DSS,which was inhibited by TMP.

    2012 02 v.25 [Abstract][OnlineView][Download 143K]

  • Determination of quality control standard for detection kit for vascular endothelial growth factor

    LI Shu-xiang*,WANG Xin-yi,LIU Jing,ZHOU Ya,XU Jing,ZOU Jian-ping(*No.3 Research Lab,National Vaccine and & Serum Institute,Beijing 100024,China)

    Objective To determine the quality control standard for detection kit for vascular endothelial growth factor(VEGF).Methods The prepared detection kit for VEGF was evaluated for linear range,minimum detection limit,precision,accuracy and stability,based on which the quality control standard was determined.Results The linear range and minimum detection limit of the prepared kit were 0 ～ 400 and 0 pg/ml respectively.The intra-precision of detection results of VEGF samples at different concentrations and time points were 9.83% ～ 13.73% and 5.47% ～ 14.08% respectively.However,the inter-precision of detection results was 7.88%.The recovery rate of VEGF at various concentrations was 94% ～105%.The quality control standard for the kit was determined as follows: both the intra-and inter-precisions were less than 15%,and the recovery rate was 90% ～ 110%.After storage at 4℃ for 6 months,all the indexes of the kit met the determined standard.Conclusion The quality control standard for detection kit for VEGF was determined,which laid a foundation of further application of the kit in clinic.

    2012 02 v.25 [Abstract][OnlineView][Download 143K]

  • Preparation and preliminary application of monoclonal antibody against gB protein of bovine rhinotracheitis virus

    WANG Bei-lei*,MENG Ri-zeng,WANG Wei,QIAN Ai-dong(*College Animal Science & Technology,Jilin Agriculture University,Changchun 130118,China)

    Objective To prepare the monoclonal antibody against gB protein of infections bovine rhinotracheitis virus(IBRV) and develop a double antibody sandwich ELISA method.Methods BALB/c mice were immunized with recombinant IBRV gB protein to prepare monoclonal antibody by hybridoma technique,and rabbits were immunized with whole IBRV to prepare polyclonal antibody.The working concentrations of prepared monoclonal and polyclonal antibodies were optimized by block titration,based on which a double antibody sandwich ELISA method was developed and verified for sensitivity and specificity.Results One hybridoma cell strains stably secreting monoclonal antibody against IBRV gB,named as 2C4,was obtained.The optimal working concentrations of polyclonal and monoclonal antibodies were 2.620 9 and 2.634 1 μg/ml respectively,and the optimal dilution of the secondary antibody was 1 ∶ 30 000.All the determination results of 100 IBRV-positive samples by the developed double antibody sandwich ELISA method were positive,indicating a coincidence rate of 100%.However,all the determination results of bovine foot-and-mouth disease virus,bovine epidemic fever virus and bovine parainfluenza virus type 3 were negative.Conclusion The monoclonal antibody against IBRV gB was successfully prepared,and a double antibody sandwich ELISA method was developed,which might be used for the diagnosis and epidemic investigation of bovine rhinotracheitis.

    2012 02 v.25 [Abstract][OnlineView][Download 148K]

  • Genetic character of wild measles virus causing an accidental case after immunization with live attenuated measles vaccine

    ZHANG Fan*,ZHOU Jian-hui,CHEN Chao,XU Xin,WANG Shuang,CHANG Xin,WEI Lei-lei,YU Jia-dong(*Lishu County Center for Disease Control and Prevention,Lishu 136500,Jilin Province,China)

    Objective To analyze the genetic character of measles virus causing a measles-like case on day 9 after immunization with live attenuated measles vaccine so as to confirm the case as vaccine-associated one or accidental one caused by wild measles virus.Methods Nucleic acids were extracted from the throat swab of the patient and sequenced after amplification of 450 nucleotides at C-terminus of measles virus nucleoprotein(NP) by RT-PCR,based on which the genetic relationship as well as homologies of nucleotides and amino acids of the strain to the representational strains of wild measles virus strains of all the 24 genotypes and Chinese vaccine strain(Shanghai-191) were analyzed.Results The virus causing the case was wild measles virus of subgenotype H1a,of which the homologies of nucleotides and amino acids were 97.5% ～ 99.5% and 96.6% respectively to those of the representational strain of wild measles virus of genotype H1 epidemic in China,while were only 91.2% and 86.7% respectively to those of Shanghai-191 strain.Conclusion The measles-like case appeared after immunization with live attenuated measles vaccine was caused by accidental infection with wild measles virus of genotype H1,but not measles vaccine-associated.

    2012 02 v.25 [Abstract][OnlineView][Download 313K]

  • Changes of monocyte chemoattractant protein-2 and IL-12 levels in peripheral blood of recipients of allogeneic hemapoietic stem cell transplantation and their correlation with acute graft versus host disease

    FEI Xiao-li,LIU Lin(Department of Hematology,Chongqing Medical University,Chongqing 400016,China)

    Objective To investigate the changes of monocyte chemoattractant protein-2/C-C motif ligand 8(MCP-2/CCL8) and IL-12 levels in peripheral blood of recipients of allogeneic hemapoietic stem cell transplantation(allo-HSCT) as well as their correlation with acute graft versus host disease(aGVHD),and provide reliable indexes for early diagnosis of aGVHD in clinic.Methods Twenty recipients of allo-HSCT were served as trial group,while those of autoplastic HSCT as control group.The MCP-2 and IL-12 levels in sera were determined 14(before pre-treatment) and 1 d(after pre-treatment and before stem cell reinfusion) before transplantation,and once a week after transplantation for 8 weeks,while those of patients with aGVHD were determined twice a week after appearance of clinical symptoms.Results In both trial and control groups,no significant differences were observed between the MCP-2 and IL-12 levels in sera 14 and 1 d before transplantation(P > 0.05),or between those 7 after and 1 d before transplantation(P > 0.05).Six cases of aGVHD were observed in trial group,of which clinical symptoms appeared 16 ～ 52 d after transplantation,and the time when serum MCP-2 and IL-12 levels increased firstly as compared with those 7 d after transplantation was earlier than that when the clinical symptoms appeared.The serum MCP-2 and IL-12 levels of the patients at time of diagnosis increased significantly as compared with those 7 d after transplantation(P < 0.05).However,after anti-aGVHD therapy,the levels decreased significantly as compared with those at time of diagnosis(P < 0.05).The serum MCP-2 and IL-12 levels of patients with aGVHD increased firstly 11～ 46 d and 11 ～ 49 d after transplantation respectively,which were significantly higher than those of recipients without aGVHD in trial group and those in control group at the same period(P < 0.05).The serum MCP-2 and IL-12 levels of recipients without aGVHD in trial group at weeks 2 ～ 8 weeks after transplantation showed no significant difference with those 1 d before transplantation(P > 0.05).Conclusion MCP-2 and IL-12 were correlated with aGVHD,of which the levels changed earlier than the appearance of clinical symptoms.The determination of serum MCP-2 and IL-12 levels was helpful to the early diagnosis of aGVHD.

    2012 02 v.25 [Abstract][OnlineView][Download 201K]

  • Distribution and persistence of neutralizing antibody titer against human cytomegalovirus in plasma donors in Sichuan Province,China

    YANG Chun,LIU Lan-jun,GAO Chang-qian,LIU Rui-xi,QIN Ting-ting,RONG Xin-zong,ZHANG Hang(Chengdu Ronsen Pharmaceuticals,Co.,Ltd.Chengdu 610041,China)

    Objective To analyze the distribution and persistence of neutralizing antibody titer against human cytomegalovirus(HCMV) in plasma donors in Sichuan Province,China.Methods A total of 2 002 serum samples of health plasma donors were collected from 8 plasma stations in Sichuan Province and determined for neutralizing antibody titer by developed method,based on which the samples from 44 donors were traced for one year,and those from one donor for 4 years.Results The neutralizing antibody titers against HCMV in plasma donors in Sichuan Province were mainly distributed in 1 ∶ 16 ～ 1 ∶ 64,while the positive rate(not less than 1 ∶ 8) was 98.35%,of which 8.04% were not less than 1 ∶ 192.The neutralizing antibody titers of 44 plasma donors were stable within the past one year.However,the neutralizing antibody titers of the donors traced for 4 years were maintained at high levels.Conclusion The natural infection rate of HCMV was high in plasma donors in Sichuan Province.The HCMV neutralizing antibody titers were high and persistent in partial donors.It provided a basis for preparation of HCMV-specific IgG.

    2012 02 v.25 [Abstract][OnlineView][Download 148K]

  • Application of complex filling material consisting of autologous skin fibroblast and hair keratin in medical cosmetology

    ZHAO Juan*,LI Xi-ning,LI Xiang-jun,YANG Min,REN Li-qun(*Department of Pathophysiology,Chengde Medical College,Chengde 067000,Hebei Province,China)

    Objective To investigate the role of complex filling material consisting of autologous skin fibroblast(Fbs) and hair keratin(HK) in removal of facial wrinkles and provide an experimental basis for application of the material in medical cosmetology.Methods Fbs isolated from volunteers were subjected to primary culture by digestion method,then to complex culture with HK particles at various ratios,and determined for survival rate by trypan blue exclusion method,for proliferation activity by MTT method,and for collagenⅠ(ColⅠ) content in medium by ELISA.The autologous Fbs extracted from opisthotic skins of 10 volunteers were co-cultured with HK particles and injected intradermally into the facial wrinkles to observe the wrinkle-removal effect.Results When the ratio of HK and Fbs was 1 ∶ 2,the survival rate and proliferation activity of Fbs as well as the ColⅠ content in medium reached peak value.Four weeks after injection,the skins at injection sites of volunteers were soft and of good compliance,while no stiffness or contracture appeared,indicating good filling effect.The appearances of volunteers were improved significantly.Conclusion The complex filling material consisting of autologous skin Fbs and HK promoted the repair of facial wrinkles significantly,which provided an experimental basis for clinical application of the material.

    2012 02 v.25 [Abstract][OnlineView][Download 320K]

  • Expressions of nuclear factor-κB-p65 mRNA and matrix metalloprotease-1 in non-small cell lung cancer tissue and their correlation to tumor metastasis

    LAN Si-you,ZHANG De-fen,DENG Shu-kai,WANG Rong-li,YANG Xiao-qiong(Department of Respiratory Medicine,The Affiliated Hospital of Luzhou Medical College,Luzhou 646000,Sichuan Province,China)

    Objective To investigate the expressions of nuclear factor(NF)-κB-p65 mRNA and matrix metalloprotease-1(MMP-1) in non-small cell lung cancer(NSCLC) tissue and their correlation to the infiltration,metastasis and apoptosis of tumor.Methods Fifty specimens of NSCLC tissue and 15 specimens of normal lung tissue adjacent to tumor were collected from surgery,then determined for expressions of NF-κB-p65 mRNA and MMP-1 by RT-PCR and immunohistochemical assay respectively,and for apoptosis index by TUNEL method.Results The expression level of NF-κB-p65 mRNA was significantly higher in NSCLC tissue specimens than in normal lung tissue adjacent to tumor(P < 0.05),while in adencarcinoma tissue than in squamous carcinoma tissue(P < 0.05).However,the positive rate of MMP-1 in NSCLC tissue was significantly higher than that in normal lung tissue adjacent to tumor(P < 0.01).The expressions of NF-κB-p65 mRNA and MMP-1 were correlated to the TNM stage,differentiation,metastasis in lymph node and pathological type of NSCLC(P < 0.05 or P < 0.01),while showed no correlation to the age and sex of patients(P > 0.05).Linear correlation analysis showed correlation between the expressions of NF-κB-p65 mRNA and MMP-1 in NSCLC tissue.The cell apoptosis index was significantly lower in NSCLC tissue than in normal lung tissue adjacent to tumor(P < 0.05),and was negatively correlated to the expression level of NF-κB-p65 mRNA(r =-0.547,P < 0.001).Conclusion In NSCLC tissue,the expression of NF-κB-p65 mRNA was correlated to that of MMP-1,and was negatively correlated to cell apoptosis index.NF-κB-p65 regulated the expression of MMP-1 protein by a certain route,and both NF-κB and MMP-1 involved in the onset and progress of tumor,in which NF-κB-p65 played an antiapoptotic role.

    2012 02 v.25 [Abstract][OnlineView][Download 299K]

  • Curative effect of interferon γ on bronchiolitis

    WANG Min*,LIANG Hang,WANG Li-na,MENG Fan-zheng(*Department of Otorhinolaryngology,Jilin City Central Hospital,Jilin 132011,Jilin Province,China)

    Objective To observe the curative effect of interferon γ(IFNγ) on bronchiolitis.Methods Eighty children meting the standard for diagnosis of bronchiolitis were randomly divided into treatment and control groups,40 for each.The children in two groups received conventional comprehensive therapy,based on which those in treatment group were further treated with IFNγ,once a day for 5 d,then observed for the time when clinical symptoms and physical signs disappeared,and determined for immunological indexes.Results Compared with those of control group,the total effective rate of treatment group increased significantly(P < 0.01),while the time when clinical symptoms and physical signs disappeared was early(P < 0.01 or P < 0.05),the serum IgE and IL-4 levels decreased significantly(P < 0.01),and the IFNγ level increased significantly(P < 0.01).Conclusion IFNγ shortened the course of bronchiolitis significantly,increased the cure rate and relieved the organic immune reaction.

    2012 02 v.25 [Abstract][OnlineView][Download 148K]

  • Preparation of gelatin microspheres

    XU Jun*,CHEN Zi-yang,SHAN Peng,CUI Ying-jie,MA Zhan-shan(*Changchun Institute of Biological Products Co.,Ltd.,Changchun 130062,China)

    Objective To preliminarily develop a procedure for preparation of interferon gelatin microspheres(IFN-GMS) and investigate their characteristics.Methods IFN-GMS were prepared,based on which the condition for preparation,such as gelatin concentration,pH value and sodium sulfate concentration,was optimized according to the status of microspheres including homogeneity,formation rate,quantity of debris as well as adhesion.The prepared IFN-GMS were determined for IFNα2b activity,based on which the encapsulation rate was calculated.Results The optimal temperature,gelatin concentration,pH value and sodium sulfate conentration for preparation of IFN-GMS were 50℃,40 g/L,3.4 or 3.6 and 300 g/L respectively.The microspheres were at a mean diameter of 18 μm,more than 70% of which were at diameters of 15 ～ 20 μm.The encapsulation rate was more than 85%.Conclusion The IFN-GMS may be used for preparation of controlled-released injection.

    2012 02 v.25 [Abstract][OnlineView][Download 192K]

  • Preparation and identification of complete chloramphenicol antigen

    WANG Ying*,AN Yu,ZHANG Dong-jie,YAO Di,LIU Zeng-shan(*Food College of Heilongjiang Agriculture University,Daqing 163319,Heilongjiang Province,China)

    Objective To prepare and identify complete chloramphenicol(CAP) antigen.Methods Immunizing antigen CAP-BSA and detection antigen CAP-OVA were prepared by diazotization,of which the coupling effects were evaluated agarose gel electrophoresis and SDS-PAGE.The molecular conjugate ratios of CAP to BSA and OVA were determined by ultraviolet scanning,while the concentration of coupled protein by BCA method.Mice were immunized i.p.with CAP-BSA and determined for serum antibody titer by indirect ELISA.Results Agarose gel electrophoresis and SDS-PAGE proved that both CAP-BSA and CAP-OVA were coupled successfully.The molecule conjugate ratios of CAP to BSA and OVA were 28︰1 and 21︰1,while the protein concentrations of CAP-BSA and CAP-OVA were 3.16 and 2.28 mg/ml,respectively.CAP-BSA stimulated high antibody titer in mice.Conclusion The completete antigens of CAP were successfully prepared,which laid a foundation of further preparation of monoclonal antibodies with high affinity and specificity.

    2012 02 v.25 [Abstract][OnlineView][Download 296K]

  • Development and preliminary application of indirect ELISA method for fibrin glue antibody

    CAI Yang,REN Zhen,HE Xue-jun,WU Hua,QIAO Hong-qun(College of Pharmacy,Nanjing University of Technology,Nanjing 210009,China)

    Objective To develop and preliminarily apply an indirect ELISA method for fibrin glue antibody.Methods An indirect ELISA method for fibrin glue antibody was developed using fibrin glue as coating antigen,of which the reaction condition was optimized,and precision and specificity were verfied.The serum antibody of rabbits immunized with fibrin glue was determined by the developed method.Results The reaction condition for the developed indirect ELISA method was optimized as follows: microtiter plate was irradiated vertically with UV light for 20 min,then coated with fibrin glue at a concentration of 10 μg/ml,diluted with 0.05 mol/L bicarbonate solution(pH 9.6) as coating buffer solution,at 4℃ overnight;the PBS containing 10% fetal bovine serum was served as blocking solution,while 0.1% PBST as antibody diluent;the optimal dilutions of polyclonal antibody against fibrin glue and HRP-labeled goat anti-rabbit IgG were 1 ∶ 2 500 and 1 ∶ 4 000 respectively.The optimal temperature and time for reaction were 37℃ and 1 h,while those for substrate coloration were 37℃ and 15 min,respectively.The sulfuric acid at a concentration of 2 mol/L was served as stop solution.The method showed high precision and specificity.The antibodies in sera of rabbits 1,2,4 and 6 weeks after immunization were determined,and the result showed that the serum antibody level increased significantly 2 weeks while started to decrease 6 weeks after immunization.Conclusion An indirect ELISA method for fibrin glue antibody was successfully developed,which might be used for the determination of fibrin glue antidoy in immune sera of rabbits.

    2012 02 v.25 [Abstract][OnlineView][Download 165K]

  • Application of hollow fiber ultrafiltration in purification of bulk of acellular pertussis vaccine

    Hu Ye-qin,Zeng Kai,Gong Jing,Zhang Bo,Chen Jing,Xue Hong-gang,Li Xin-guo,Xiang Mei-juan(Wuhan Institute of Biologic Products Co.,Ltd.,Wuhan 430060,China)

    Objective To purify detoxified acellular pertussis antigen by hollow fiber ultrafiltration.Methods The acellular pertussis antigen after detoxification was purified by hollow fiber ultrafiltration,of which the purity,recovery rate and titer were compared with those purified by dialysis.Results Hollow fiber ultrafiltration was highly effective in removal of detoxifying agent and pigment in acellular pertussis antigen solution,which was completed within 3 h.The recovery rate of purified bulk was 92.1%,while the titer(11.166 IU/ml) was higher than that purified by dialysis(9.211 IU/ml).Conclusion Hollow fiber ultrafitration may be used for purification of acellular pertussis antigen instead of dialysis.

    2012 02 v.25 [Abstract][OnlineView][Download 117K]

  • Application of whole-cell subtractive panning in phage display library screening

    WU Hong-zhen,ZHANG Lin-bo(Innovation Laboratory of Biopharmaceuticals,College of Life Science,Jilin Agriculture University,Changchun 130118,China)

    Whole-cell subtractive panning is a screening technique developed based on whole-cell screening in recent years,which performs subtractive panning on phage display library by using couple cells,i.e.two states of cells.It is mainly used for screening of novel antigenic epitopes,receptors,ligands,novel vaccines,the targeted active peptides of tumor cells,targeted gene vectors,and stimulants or antagonists of receptors or enzyme etc.The principle,advantages,disadvantages and optimization of the technique as well as its application in phage display library screening are reviewed in this paper.

    2012 02 v.25 [Abstract][OnlineView][Download 160K]

  • Development of laboratory detection technique for HCV and its suitability in various courses

    LI Sheng-tao*,XIA Xue-shan,DUAN Hai-ping(*Life Science and Technology Institute of Molecular Virology Laboratory,Kunming University of Science and Technology,Kunming 650224,China)

    Hepatitis C(HC) seriously threatens the health of humans.Hepatitis C virus(HCV) infection shows a complex course,including acute phase,chronic phase until development of cirrhosis and liver cancer,as well as chronic latent infection.Accurate and reliable laboratory detection technique is the basis of prevention,control and prognosis of the disease as well as development of effective drugs.The laboratory detection technique for HCV is developing rapidly,of which the sensitivity and specificity are significantly improved.However,the scope of suitability of laboratory detection technique is variable according to the various courses.This paper reviews the development of laboratory detection technique for HCV in recent years as well as its suitability in various courses.

    2012 02 v.25 [Abstract][OnlineView][Download 191K]


@2012 Editorial Department of "Chinese Journal of Biologicals"