2010年08期目次

  • Effect of Bone Mesenchymal Stem Cells on Differentiation of Rat Glioma C6 Cell Line

    WANG Xiao-dong, MU Li-li, JI Yu-huan, SUN Bo, WANG Guang-you, HAN Zhi-juan, LIU Ting-ting, LI Hu-lun, WANG Jing-hua (Emergency Department, The Second Affiliated Hospital of Harbin Medical University, Harbin 150086, China)

    Objective To investigate the effect of bone mesenchymal stem cells(BMSCs)on the differentiation of rat glioma C6 cell line and the relevant mechanism. Methods BMSCs and C6 cells were co-cultured in Transwell, then determined for proliferation level of C6 cells by cell counting, for cell cycle of C6 cells by flow cytometry, for expressions of vimentin and glial fibrillary acidic protein(GFAP)by IFA and immunohistochemical assay respectively, and for activity of glutamine synthetase(GS)by biochemical assay. Results BMSCs showed inhibitory effect on the proliferation of C6 cells. The arrest of C6 cells at G0 / G1 phase was observed 72 h after co-culture with BMSCs. The expression of GFAP in C6 cells co-cultured with BMSCs was up-regulated significantly, while that of vimentin was down-regulated. In addition, the distributions and arrangements of GFAP and vimentin as markers of differentiation in cells changed. The GS activity in C6 cells co-cultured with BMSCs increased. Conclusion BMSCs induced the differentiation of C6 cells to mature astrocytes, which might be associated with some cytokines secreted by BMSCs.

    2010 08 v.23 [Abstract][OnlineView][Download 463K]

  • Preliminary Evaluation on General Toxicity of BCG-CpG-DNA

    ZHAO Ai-hua, QIAO Lai-yan, JIA Shu-zhen, KOU Li-jie, WANG Wen, LI Feng-xiang, WANG Guo-zhi(National Institute for the Control of Pharmaceutical and Biological Products, Beijing 100050, China)

    Objective To observe the acute and chronic toxicities of BCG-CpG-DNA in mice and evaluate the general toxicity. Methods Eighty KM mice were divided into one control and three test groups, 20 for each. The mice in control group were s. c. injected once with physiological saline, while those in three test groups once with 100, 250 and 500 μg of BCG-CpG-DNA for acute toxicity test. However, another 80 mice were grouped by the same method and injected for 7 times on days 0, 3, 7, 10, 14, 21 and 28 respectively for chronic toxicity test. The growth, bodyweight and pathological change of mice in acute toxicity test were observed. The physical signs, local irritations and body weights of mice were observed once a week until 3 weeks after the last injection. The haematological and blood biochemical indexes, main organ coefficients as well as anti-dsDNA antibody levels of mice were determined 3 d and 3 weeks after the last injection. Results In acute toxicity test, all the mice survived with bodyweights gained and showed no toxic reaction. In chronic toxicity test, no abnormalities in the bodyweights or behaviors or local irritation of mice were observed. Repeat administration with BCG-CpG-DNA increased the immune cell counts of mice. The spleen coefficients of mice in various test groups 3 d after the last injection were significantly higher than those in control groups (P < 0. 05), which recovered to the normal level 3 weeks later. The anti-dsDNA antibody levels in sera of mice in various test groups showed no significant difference with those in control group, both of which were less than a cutoff value of 25 U / ml. Conclusion BCG-CpG-DNA mainly affected the immune cells and immune organs of mice, while showed no significantly acute or chronic toxicities, indicating good safety.

    2010 08 v.23 [Abstract][OnlineView][Download 229K]

  • Effect of S-Adenosyl-L-Methionine on Proliferation and Migration of Human Liver Cancer HepG2 Cells and Expression of Cancer Gene C-myc

    ZHOU Wei, BI Yang, FENG Tao, WEN Wei, LU Xiao-juan, ZHAO Ying-ze, ZHANG Ting (Molecular Medicine and Cancer Research Center, College of Basic Medicine, Chongqing Medical University, Chongqing 400016, China)

    Objective To investigate the effect of S-adenosyl-L-methionine(SAMe)on the proliferation and migration of human liver cancer HepG2 cells as well as the expression of cancer gene C-myc, and lay a foundation of further study on the role of SAMe in therapy of liver cancer. Methods HepG2 cells were cultured in vitro and treated with SAMe at various concentrations for various days, and the effect on cell proliferation was determined by MTT method. The effect of SAMe at optimal concentration on the migration of HepG2 cells was determined by cell streak. The effects of SAMe on the transcription of C-myc gene as well as location and expression of protein in HepG2 cells were determined by RT-PCR, immunocytochemical method and Western blot respectively. Results The inhibition rate of HepG2 cells after treatment with SAMe at a final concentration of 15. 0 μmol / L for 5 d reached 54. 6%, and the inhibitory effect was time- and dose-dependent. The migration ability of HepG2 cells after treatment with 15. 0 μmol / L SAMe decreased significantly, with a speed for healing of streak equivalent to 76. 78% of that in control group. Both the transcription level of C-myc gene and expression of C-myc protein in HepG2 cells treated with SAMe decreased significantly, while the location of protein showed no significant change. Conclusion SAMe inhibited the proliferation and migration of HepG2 cells by decreasing the expression of C-myc gene, which provided a new pathway for development of drugs for liver cancer.

    2010 08 v.23 [Abstract][OnlineView][Download 401K]

  • Role of Uncoupling Protein-2 in Insulin-resistance Induced by Palmic Acid in HepG2 Cells and Its Relationship to Nuclear Factor-κB

    WANG Li-juan, GUAN Xiao-qin, LIU Lin, TANG Yuan-ting, ZHU Liang-rong(Department of Pathology, Molecular Medicine and Cancer Research Center, Chongqing Medical University, Chongqing 400016, China)

    Objective To investigate the role of uncoupling protein-2(UCP-2)in inulin-resistance induced by palmic acid(PA) in HepG2 cells and its relationship to nuclear factor-κB(NF-κB). Methods HepG2 cells were divided into normal control, PA, high insulin(HI)and PA + Genipin groups. The cells in normal control groups were cultured in RPMI1640 medium containing 10 calf serum, while those in PA, HI and PA + Genipin groups in the media added with 0. 25 mmol / L PA, 100 nmol / L insulin and 0. 25 mmol / L PA + 10 μmol / L Genipin respectively, for 24 h, then stimulated with 100 nmol / L insulin. The glucose, MDA, SOD, ALT, AST, GGT and TG concentrations in media were determined, the fatty degeneration of cells was observed by oil red O staining, the change of mitochondrial membrane potential was analyzed by flow cytometry, and the expression levels of IRS-2, UCP-2 and NF-κB were determined by RT-PCR and Western blot. Results The glucose contents in media of PA and HI groups 12 h after stimulation with insulin showed no significant difference (P > 0. 05). The glucose, ALT, AST, MDA, GGT and TG contents as well as the expression levels of UCP-2 and NF-κB were significantly higher in PA group than in normal control and PA + Genipin groups (P < 0. 05), while showed no significant difference in PA + Genipin and normal control groups(P > 0. 05). The mitochondrial membrane potential as well as SOD and IRS-2 levels were significantly lower in PA group than in normal control and PA + Genipin groups (P < 0. 05), while showed no significant difference in PA + Genipin and normal control groups (P > 0. 05). Conclusion UCP-2 played an important role in the inulin-resistance induced by PA in HepG2 cells, of which the mechanism might be related to NF-κB.

    2010 08 v.23 [Abstract][OnlineView][Download 368K]

  • Prokaryotic Expression and Purification of Acid-resistance-associated Two Component System Protein ArsS from Helicobacter pylori

    ZHANG Xiao-li, ZHANG Jin-yong, ZOU Quan-ming (Department of Clinical Microbiology and Immunology, The Third Military Medical University, Chongqing Engineering Technology Research Center of Biopharmaceuticals, Chongqing 400038, China)

    Objective To express the acid-resistance-associated two component system protein ArsS of Helicobacter pylori in prokaryotic cells, purify the expressed product and lay a foundation of further study on its function as well as its role in acid-resistance mechanism of H. pylori. Methods ArsS gene was amplified by PCR using the genome of H. pylori isolate 26695 as a template and inserted into vector pET-22b(+). The constructed recombinant plasmid pET-22b(+)-ArsS was transformed to E. coli BL21(DE3)for expression under induction of 0. 5 mmol / L IPTG at 25℃. The expressed product was purified by affinity chromatography and molecular sieve chromatography. Results Both restriction analysis and sequencing proved that recombinant plasmid pET-22b(+)-ArsS was constructed correctly. The expressed product, in a soluble form, contained more than 30% of total somatic protein, and was effectively purified by molecular sieve chromatography using a buffer containing 150 mmol / L sodium chloride. The purified protein, at a concentration of about 10 mg / ml, reached a purity of more than 95%. Conclusion ArsS protein was successfully expressed in prokaryotic cells and purified.

    2010 08 v.23 [Abstract][OnlineView][Download 248K]

  • Differential Expression of Heme Oxygenase-1 in PC12 Cell Model of Parkinson Disease Induced by Proteasome Inhibitor

    LIU Tao, JIN Ying-hua, ZHANG Yu, CHANG Ming, WANG Dan-ping, HU Lin-sen (Department of Neurology, First Hospital, Jilin University, Changchun 130021, China)

    Objective To investigate the differential expression of heme oxygenase-1(HO-1)in PC12 cell model of Parkinson disease (PD)induced by proteasome inhibitor (PSI)and provide theoretical basis for further study on pathogenic mechanism of PD. Methods PC12 cell model of PD was established by adding PSI to the cultured PC12 cells to a final concentration of 10 μmol / L, using the cells added with DMSO to a final concentration of 10 μmol / L as control, and identified by HE, AO & EB and α-SYN staining. Proteins were extracted from the PC12 cells 48 h after treatment with PSI to obtain differential protein spots by DIGE and identify differential proteins by MALDI-TOF-Pro MS. Results Acidophilic Lewy-like bodies were formed in the PC12 cells 48 h after treatment with PSI, and the apoptosis rate of cells reached(24. 74 ± 4. 55)%. The expression level of HO-1 in PC12 cells treated with PSI increased significantly as compared with those treated with DMSO. Conclusion Oxidative stress plays an important role in the pathogenesis of PD induced by functional disorder of ubiquitin-proteasome system(UPS).

    2010 08 v.23 [Abstract][OnlineView][Download 459K]

  • Effect of Diabetic Nephropathy-associated microRNA on Mesangial Cell Proliferation Induced by High Glucose and Relevant Mechanism

    ZHANG Zheng, PENG Hui-min, XU Xiao-ming, CHEN Tao, HE Xiao-yan(Department of Cell Biology and Genetics, College of Basic Medicine, Molecular Medicine and Tumor Research Center, Chongqing Medical University, Chongqing 400016, China)

    Objective To investigate the effect of diabetic nephropathy(DN)-associated microRNA miR-21 on the growth and proliferation of mesangial cells at high glucose concentration as well as the effect of high expression of miR-21 on PTEN / PI3K signal pathway. Methods Mouse mesangial cells were cultured at high glucose concentration to mimic diabetes mellitus, and transfected with eukaryotic expression vector pGenesil-miR-21 in mediation of Lipofectamine 2000. The cell clones for stable expression were screened with G418. The expression level of miR-21 in transfected cells was determined by real-time RT-PCR, and the cell prolifera- tion by MTT method. The expressions of PTEN, phospho-Akt (Ser473)and PI3K p85α proteins, the key molecules of PTEN / PI3K signal pathway, were determined by immunocytochemical method. Results The miR-21 was expressed highly and stably in the screened mesangial cells. The highly expressed miR-21 significantly inhibited the proliferation of mesangial cells and down-regulated the expression of PTEN(P < 0. 05), while significantly up-regulated the expressions of phospho-Akt(Ser473)and PI3K p85α(P < 0. 05). Conclusion The miR-21 may increase the expression levels of phospho-Akt(Ser473)and PI3K p85α proteins by specific inhibiting the expression of PTEN protein, thus inhibit the proliferation of mesangial cells, which may be a novel potential target for prevention and therapy of DN.

    2010 08 v.23 [Abstract][OnlineView][Download 289K]

  • Relationship of Retained Fetal Membranes of Cows to Expressions of iNOS, eNOS and MMP-2 in Placenta

    FU Shi-xin, ZHANG Li, QI Chang-xue, LUO Chun-hai, LI Peng, XIA Cheng(College of Animal Sciences and Technology, Heilongjiang August First Land Reclamation University, Daqing 163319, Heilongjiang Province, China)

    Objective To analyze the retained retention of fetal membrane(RFM) of cows to the expressions of induction nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS)and matrix metallopeptidase-2 (MMP-2)in placenta as well as the molecular mechanism of onset of RFM. Methods The iNOS, eNOS and MMP-2 genes were amplified by RT-PCR from the placentas of cows with no RFM (NRFM)and with RFM separately and cloned, using bovine β-actin as an internal reference, and the expression levels were determined by fluorescent quantitative PCR. Results The iNOS, eNOS and MMP-2 genes were cloned, of which the homologies of nucleotide sequences were 100% to those reported in GenBank. The transcription level of iNOS mRNA in the placenta of cows in RFM group was significantly higher, while those of eNOS and MMP-2 mRNAs were significantly lower than those in NRFM group(P < 0. 05). Conclusion The transcription levels of iNOS, eNOS and MMP-2 mRNAs were closely related to the onset of RFM.

    2010 08 v.23 [Abstract][OnlineView][Download 406K]

  • Prokaryotic Expression and Antimicrobial Activity of Human Lysozyme-Tachyplesins Fusion Protein

    OUYANG Ping, GAO Yu, LEI Lian-cheng, YIN Li-zi, JIANG Li-na, LV Shuang, FENG Xin, HAN Wen-yu(College of Animal Science and Veterinary Medicine, Jilin University, Changchun 130062, China)

    Objective To express the fusion protein of human lysozyme(hLYZ)and tachyplesins, an antimicrobial peptide, in prokaryotic cells, and determine its antimicrobial activity. Methods The gene and linker of tachyplesins were synthesized and fused with the hLYZ gene cut from plasmid pMD18-T-hLYZ, then cloned into prokaryotic expression vector pGEX-4T-1 with GST labeling. The constructed recombinant plasmid pGEX-4T-1-hLYZ-tachyplesins was transformed to E. coli BL21(DE3)for expression under induction of IPTG, and the condition for expression was optimized. The expressed fusion protein was purified by affinity chromatography and determined for antimicrobial activity. Results PCR, restriction analysis and sequencing proved that recombinant plasmid pGEX4T-1-hLYZ-tachyplesins was constructed correctly. The expressed product showed a band with relative molecular mass of about 44 000 on SDS-PAGE profile. The fusion protein was expressed mainly in a soluble form after induction with 0. 8 mmol / L IPTG at 25℃ for 6 h. The purified fusion protein reached a purity of more than 90% and showed a certain inhibitory effect on both Staphylococcus aureus and E. coli. Conclusion The hLYZ-tachyplesins fusion protein was successfully expressed in prokaryotic cells. The purified fusion protein showed a certain antimicrobial activity.

    2010 08 v.23 [Abstract][OnlineView][Download 489K]

  • Construction and Expression of Recombinant Human Ribonuclease Inhibitor Adeno-associated Virus

    CHU Ying-hao, OUYANG Xi, HE Xiao-yan, CHEN Jun-xia, GAO Juan, ZHU Jun(Department of Cellular Biology and Genetics, Chongqing Medical University, Chongqing 400016, China)

    Objective To construct recombinant adeno-associated virus(AAV)carrying full-length of human ribonuclease inhibitor(RI)gene and express in bladder cancer T24 cells. Methods Total RNA was extracted from human umbilical vein endothelial cells ECV30 by Trizol method for amplification of RI gene by RT-PCR. The amplified RI gene was cloned into vector pUCm-T, then subcloned to vector pAAV-IRES-hrGFP. The constructed recombinant plasmid pAAV-IRES-hrGFP-RI was co-transfected with plasmids pAAV-RC and pAAV-Helper to HEK293 cells from which RI-AAV was packaged, amplified and determined for infectious titer, then transfected to T24 cells. The transcription level of RI mRNA was determined by RT-PCR, and the expression level of RI protein by Western blot. Results Both restriction analysis and sequencing proved that recombinant plasmid pAAV-IRES-hrGFP-RI was constructed correctly. The infectious titer of RI-AAV was 1. 1 × 1010 U / ml. Both the transcription level of RI mRNA and the expression level of RI protein in T24 cells transfected with RI-AAV were significantly higher than those in the cells untransfected and transfected with empty AAV(P < 0. 05). Conclusion The recombinant AAV carrying human RI gene was successfully constructed, which laid a foundation of further study on the role of RI in invasion and metabasis of bladder cancer cells.

    2010 08 v.23 [Abstract][OnlineView][Download 289K]

  • Effect of Cardiopulmonary Bypass on Glucose Transporter 4 in Skeletal Muscle and Glucose Transporter 2 in Islets of Rabbits

    TONG Shan-shan, MIN Su, WEI Ke, ZHANG Guang-xin(Department of Anesthesiology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China)

    Objective To observe the effect of cardiopulmonary bypass(CPB) on the glucose transporter 4(GLUT4)in skeletal muscle and GLUT 2 in islets of rabbits and investigate the potential mechanism of hyperglycaemia in CPB. Methods Twenty-four New Zealand rabbits were divided into normal control(N)and CPB(C)groups, 12 for each. Rabbit model of CPB was established by using the rabbits in C group. The arterial blood samples of rabbits in the two groups were collected immediately after narcosis(T1), immediately after aortic cross-clamping(T2)as well as 5(T3), 35(T4)and 75 min(T5)after reperfusion and determined for blood glucose by oxidase test and for insulin by radioimmunoassay. The transposition expressions of total GLUT4 of skeletal muscle and GLUT4 on cell membrane 5 and 75 min as well as expression of GLUT2 in islet cells 75 min after reperfusion were determined by im- munohistochemical assay. Results Both the blood glucose and insulin levels at T2～T5 of the two groups increased significantly (P < 0. 05)as compared with those at T1, and were significantly higher in group C than in group N (P < 0. 05). Both the transposition expressions of total GLUT4 of skeletal muscle and GLUT4 on cell membrane of rabbits in group C 5 and 75 min after reperfusion were down-regulated significantly(P < 0. 05), while the expression of GLUT2 in islet cells 75 min after reperfusion was up-regulated significantly(P < 0. 05). However, both the transposition expression levels of total GLUT4 of skeletal muscle and GLUT4 on cell membrane of rabbits in group C 75 min after reperfusion were significantly higher than those 5 min after reperfusion. Conclusion The hyperglycaemia in CPB of rabbits might be associated with the down-regulation of expression and disturbance of transposition of GLUT4 in skeletal muscle. Though the up-regulation of GLUT2 expression in islet cells might increase the secretion of insulin, the high blood glucose level could not be compensated completely by the increased insulin.

    2010 08 v.23 [Abstract][OnlineView][Download 222K]

  • Effect of Modified RPMI1640 Medium on Proliferation of CIK Cells

    MENG Ming-yao, XIE Yan-hua, LIU Hong-wei, LIU Yun-hong, HOU Zong-liu(Central Laboratory, Yan'an Hospital of Kunming Medical College, Kunming 650031, China)

    Objective To investigate the effect of modified RPMI1640 medium on the in vitro proliferation of CIK cells and provide CIK cells at high quality and quantity for clinical therapy. Methods CIK cells were isolated by using Ficoll lymphocyte separating medium, then cultured in traditional and modified RPMI1640 media respectively. On days 0, 3, 6, 9, 12, 15 and 18 after culture, the cells were analyzed for proliferation level by counting with trypan blue exclusion staining, for phenotype by flow cytometry, and for in vitro cytotoxic activity by MTT method. Results The proliferation fold of CIK cells on day 12 after culture in modified RPMI1640 medium was significantly higher than that in traditional medium (P < 0. 05), while the percentage of CD3+ CD56+ cells was more than 33%. Both the cytotoxic activities of CIK cells on day 15 after culture in modified medium to BGC-823 and SPC-A-1 cells were higher than those in traditional medium. Conclusion Compared with traditional RPMI1640 medium, the modified RPMI1640 medium promoted the proliferation of CIK cells effectively, which provided an experimental basis for clinical application of CIK cells.

    2010 08 v.23 [Abstract][OnlineView][Download 149K]

  • Screening of Host Bacterial Strain and Optimization of Fermentation Condition for Production of DNA Vaccine against Schistosoma japonicum

    ZHU Lu, LIU Hai-feng, LONG Quan-ke, LU Ming-bo, YU Long-jiang(Key Laboratory of Molecular Biophysics of Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China)

    Objective To screen the host bacterial strain of bivalent DNA vaccine against Schistosoma japonicum and optimize the condition for fermentation of the strain. Methods The host bacterial strain as well as the carbon source, nitrogen source and phosphate of fermentation medium were optimized by synthetic evaluation of dry cell weight, plasmid DNA yield and stability of plasmids in subcultured recombinant strain. Fermentation medium was further optimized by central combination design response surface test, then verified. Results E. coli DH5α was screened as the host bacterial strain of DNA vaccine against Schistosoma japonicum. The optimal fermentation medium consisted of(m / v)1. 0% sodium chloride, 1. 0% yeast Extract, 0. 3% phosphate in which the molar ratio of dipotassium hydrogen phosphate to potsssium dihydrogen phosphate was 1 ∶ 4, 1. 68% glycerol, 2. 28% beef juice and 4. 21% bean juice. Under the optimized fermentation condition, the dry cell weight increased from 1. 57 mg / ml to 5. 68 mg / ml, while the plasmid DNA yield from 3. 94 μg / ml to 36. 52 μg / ml. Conclusion The host bacterial strain of DNA vaccine against Schistosoma japonicum was screened, and the fermentation condition was optimized, by which the plasmid DNA yield increased significantly, indicating that the screened host bacterial strain and the optimized fermentation condition were suitable for large scale production of DNA vaccine against Schistosoma japonicum.

    2010 08 v.23 [Abstract][OnlineView][Download 542K]

  • Immune Response Induced by Rabies Vaccine Containing Poly IC Adjuvant in Mice

    LI Mao-guang, YU Yong-xin, LI Jia, LIU Jing-hua, LIU Xin-yu, XU Hong-shan, TANG Jian-rong, DONG Guan-mu (National Institute for the Control of Pharmaceutical and Biological Products, Beijing 100050, China)

    Objective To observe the immune response induced by rabies vaccine containing Poly IC adjuvant in mice. Methods BALB / c mice were divided into three test and two control groups, 15 for each. The mice in three test groups were injected i.p. with diluted hamster kidney cells-based rabies vaccine containing Poly IC adjuvant (PHKCV+P), hamster kidney cells-based rabies vaccine for human use(PHKCV)and national rabies vaccine reference(R)respectively, once on day 0 or twice on days 0 and 7. The mice in one control group(P)were injected with Poly IC adjuvant, while those in the other control group(N)were untreated. Five mice in each group were killed on days 7 and 14 respectively, and their sera were separated for determination of neutralizing antibody. Meanwhile, the spleens were collected for determination of secreted IFNγ levels by ELISPOT method. The rest ten mice in each group were challenged i.c. with CVS strain on days 7 and 14 to observe the protective effect of vaccine. Results The serum neutralizing antibody levels of mice immunized twice with PHKCV+P were significantly higher than those with PHKCV. However, both the specific IFNγ spot formation cell (SFC)counts of mice immunized once and twice with PHKCV+P were significantly higher than those in other groups, while the protective effects were superior to that in PHKCV group. Conclusion Rabies vaccine containing Poly IC adjuvant decreased the amount of antigen used, while enhanced the cellular and humoral immune responses especially early cellular immunity, thus induced higher protective efficacy as compared with traditional rabies vaccine.

    2010 08 v.23 [Abstract][OnlineView][Download 289K]

  • Screening and Biological Characteristics of Vero Cell-adapted Reassortant Rotavirus LD9 Strain(G2 Type)

    KOU Gui-ying, HU Guang-hong, BAO Hong, WANG Ming-qiang, CHEN Han-quan, WANG Zhou, ZHOU Xu (Lanzhou Institute of Biological Products, Lanzhou 730046, China)

    Objective To investigate the adaptability of reassortant rotavirus LD9 strain(G2 type)in Vero cells and screen the adapted strain with high titer. Methods Three clones of reassortant rotavirus LD9 strain(G2 type), i.e. LD9-1, LD9-2 and LD9-3, were inoculated into Vero cells at MOIs of 0. 05, 0. 10, 0. 15 and 0. 20 separately for subculture, from which one Vero cell-adapted strain was screened, and its MOI was optimized. The adapted strain was subcultured in Vero cells for 15 passages then determined for virus titer and nucleic acid band patterns of genome, from which VP7 gene was amplified by nest RT-PCR. The adapted strain of passages 8, 10 and 13 were inoculated into Vero cells at a MOI of 0. 10 and observed for proliferation. Results The screened LD9-2 strain, inoculated into Vero cells at a MOI of 0. 10, caused obvious CPE, of which the titer decreased at first then increased with the increasing passages. The titer of passage 4 was the lowest, while those of more than passage 9 was stable at about 6. 75 lgCCID50 / ml. All the nucleic acid band patterns of the adapted strain of passages 1 ～ 15 were consistent with that of primary strain, from each of which the VP7 gene at a length of 502 bp was amplified. All the proliferation peaks of LD9-2 strain of passages 8, 10 and 13 appeared on days 7 ～ 8 after culture, with titers of 6. 0 ～ 7. 0 lgCCID50 / ml. Conclusion A candidate rotavirus vaccine strain (G2 type)with high titer, which may be stably subcultured, is obtained by adaptive culture of reassortant rotavirus LD9 strain in Vero cells.

    2010 08 v.23 [Abstract][OnlineView][Download 256K]

  • Laboratory Evaluation of Method for Determination of Neutralizing Antibody against Human Enterovirus 71

    MAO Qun-ying , HE Peng, YU Xiang, LI Nan, HAO Chun-sheng, GAO Qiang, DONG Cheng-hong, LIANG Zheng-lun, LI Feng-xiang, SHEN Xin-liang, WANG Jun-zhi(National Institute for the Control of Pharmaceutical and Biological Products, Beijing 100050, China)

    Objective To evaluate the method for determination of neutralizing antibody against human enterovirus 71 (EV71)in laboratory and provide a basis for standardization of the method. Methods The neutralizing antibody titers against EV71 in 10 human immunoglobulin, 20 healthy adult plasma and 15 high titer animal immune serum specimens were determined with 10 EV71 strains of genotypes A, B and C by 5 laboratories according to the SOP for EV71 neutralizing antibody, based on which the intra-and inter-reproducibility of the method as well as effect of various EV71 strains on determination of neutralizing antibody were analyzed. Results The fold differences in maximum and minimum(Max-Min)of determination results of neutralizing antibody titers with various EV71 strains by the same laboratory were less than 4, and the results of 3 repeat tests showed no significant difference (P > 0. 05). The fold differences in determination results with the same EV71 strain by various laboratories were also less than 4. The determination results with various EV71 strains by various laboratories were as follows: the fold differences of the Max-Min of neutralizing antibody titers of EV71 strain genotype A with other genotypes were not more than 192, while those of subtypes B3 with C4 were not more than 64, and those of subtype C4 with other strains were not more than 16. Conclusion By operation according to the unitary SOP, the method for determination of neutralizing antibody against EV71 showed satisfactory intra- and inter-reproducibility. However, various EV71 strains showed significant effect on the determination result.

    2010 08 v.23 [Abstract][OnlineView][Download 160K]

  • Analysis of Stability of Superoxide Dismutase Solution by Steady-state Fluorescence Spectroscopy

    ZHOU Jia-min, LI Chen-wen, WANG Yi-xin, YIN Zong-ning (Sichuan School of Health, Chengdu 610100, China)

    Objective To investigate the effects of various influencing factors on activity and conformation of superoxide dismutase(SOD). Methods The activity of SOD solution was determined by pyrogallol auto-oxidation rate method. The relationship between the changes of fluorescence spectrum and those of tertiary and quarternary structures of SOD in presence of various influencing factors(pH value, ultrasonication and guanidine hydrochloride as denaturant)was analyzed by steady-state fluorescence spectroscopy. Results The stability of SOD increased with the decreasing pH value. The pH value at a range of 2. 2 ～ 7. 0 was linearly related to specific activity of SOD. With the decreasing pH value, the maximum emission wavelength of general fluorescence showed a blue shift from 336 nm to 325 nm. The increased times of ultrasonication caused the blue shift of synchronous spectrum bands and the decrease of SOD activity. The SOD activity decreased gradually with the increasing guanidine hydrochloride concentration. When the guanidine hydrochloride concentration reached 2. 1 mol / L, the maximum fluorescent emission peak showed a blue shift to 312 nm. The fluorescence intensity of SOD de-naturalized with guanidine hydrochloride increased as compared with that of natural SOD. Conclusion It is feasible to analyze the stability of SOD by steady-state fluorescence spectroscopy.

    2010 08 v.23 [Abstract][OnlineView][Download 346K]

  • Determination of Concentration of Anthrax Vaccine by Spectrophotometry

    WEI Dong, WANG Bing-xiang, LI Ke-mei, PEI Ming-yu, LI Xiao-li, WANG Guo-zhi(National Institute for the Control of Pharmaceutical and Biological Products, Beijing 100050, China)

    Objective To determine the concentration of anthrax vaccine by spectrophotometry. Methods The wavelength and linear range for spectrophotometry of anthrax vaccine were determined, based on which the stability of test samples and repro - ducibility of the method were analyzed. Results The wavelength for spectrophotometry of anthrax vaccine was determined as 600 nm. The A600 value showed a good relationship(r = 0. 999 1, df = 3, P < 0. 01)to anthrax vaccine concentration at a range of(0. 25 ～ 1. 25)× 108 bacteria / ml. The stability of test samples increased after treatment with formaldehyde. The method showed high reproducibility. Conclusion Spectrophotometry may be used for determination of anthrax vaccine concentration instead of turbidimetry or as a method for confirmation.

    2010 08 v.23 [Abstract][OnlineView][Download 183K]

  • Establishment of Method and Standard for Quality Control of Recombinant Human Monoclonal Antibody against Rabies

    WEI Jing-shuang, CHENG Li-jun, ZHAO Wei, ZHOU Xing-jun, LIU Jin-huai, JIA Qian(New Drug R&D Center, North China Pharmaceutical Corporation, Shijiazhuang 050015, China)

    Objective To establish the method and standard for quality control of recombinant human monoclonal antibody against rabies (rhRMcAb). Methods The rhRMcAb was analyzed for neutralizing activity by mouse neutralization test(MNT)and rapid fluorescent focus inhibition test(RFFIT), for purity by reduced and non-reduced SDS-PAGE and for peptide map by reduction alkylation and trypsin digestion followed by reverse phase HPLC. The amino acids at N-terminus was sequenced by amino acid sequencer after the pyroglutamate blockage at N-terminus was removed by pyroglutamate aminopeptidase. The affinity constant of rhRM-cAb with rabies virus glycoprotein was determined by surface plasmon resonance(SPR)method. Other quality indexes of rhRMcAb were determined according to the requirements in Chinese Pharmacopeia (2005 edition), based on which a standard for quality control was established. Results The activities of six batches of rhRMcAb determined by MNT and RFFIT were basically in agreement. The reduced SDS-PAGE purities of three batches of rhRMcAb bulks were more than 99. 0%. Except major antibody bands, some minor bands with both high and low relative molecular masses were also observed on the non-reduced SDS-PAGE profile of rhRMcAb. The peptide maps of three batches of bulks were consistent with that of in-house reference. The amino acid sequence at N-terminus was identical to the theoretical value. The affinity constant of rhRMcAb with rabies virus glycoprotein was 1. 86 × 108 / M. All the other quality indexes of rhRMcAb met the requirements in Chinese Pharmacopeia(2005 edition). In the established standard for quality control, the neutralizing activity of rhRMcAb was not less than 500 IU / ml, while both SDS-PAGE and HPLC purities were not less than 98. 0%. Conclusion The method and standard for quality control of rhRMcAb were established.

    2010 08 v.23 [Abstract][OnlineView][Download 363K]

  • Inhibitory Effect of Evodiamine on Proliferation of Human Colon Cancer lovo Cells

    ZHANG Chun, YANG Xue, FAN Xia, WEI Qiang, WANG Xi, ZHAO Yong, LIANG Hua-ping (Department of Pathology, The Molecular Medicine and Cancer Research Center, Chongqing Medical University, Chongqing 400016, China)

    Objective To investigate the in vitro and in vivo inhibitory effects of evodiamine(Evo)on the proliferation of human colon cancer lovo cells as well as the relevant mechanism. Methods The effects of Evo on the proliferations of lovo cells, human breast cancer MDA-MB-231 cells and human lung cancer A549 cells were determined by MTT method. The effects of Evo on the cell cycle and apoptosis of lovo cells were analyzed by flow cytometry. Nude mice bearing lovo cells were treated with Evo, based on which a tumor growth curved was plotted. The effects of Evo on the expressions of Bcl-2 and procaspase-3 in lovo cells and tumor tissue were determined by Western blot. Results Evo inhibited the proliferation of lovo cells (P < 0. 01)and promoted that of MDAMB-231 cells(P < 0. 01)significantly, while showed no significant effect on A549 cells. Evo arrested lovo cells at S phase(P < 0.01), induced the apoptosis of the cells in a time-dependent mode(P < 0. 01), and inhibited the growths of tumors in the mice bearing lovo cells (P < 0. 05). Western blot showed both in vitro and in vivo inhibitory effects of Evo on expressions of Bcl-2 and procaspase-3. Conclusion Evo may activate caspase-3 and induce cell apoptosis by inhibiting the expression of Bcl-2, thus inhibit the proliferation of lovo cells.

    2010 08 v.23 [Abstract][OnlineView][Download 337K]

  • Effect of Sodium Ferulate on Expression of Connective Tissue Growth Factor in Myocardial Tissue of Rats with Myocardial Fibrosis

    GAO Hai-cheng, GAO Hai-mei, REN Li-qun(School of Pharmacy, Jilin University, Changchun 130021, China)

    Objective To investigate the effect of sodium ferulate(SF)on the expression of connective tissue growth factor (CTGF)in myocardial tissue of rats with myocardial fibrosis(MF). Methods Wistar rat model of MF was copied by a single s.c. injection with isoproterenol (Iso)at a dosage of 15 mg / kg bodyweight. The model rats were killed at various time points and determined for transcription level of CTGF mRNA in myocardial tissue by semi-quantitative RT-PCR, and for expression level CTGF by immunohistochemical assay. Wistar rat model of MF was copied again, with synchronous intervention by injection with SF. The transcription level of CTGF mRNA and expression level of CTGF in the myocardial tissue of model rats were determined 3 weeks later. Results The transcription level of CTGF mRNA in model rats reached a peak value and was significantly higher than that in normal control group 24 h, and was still higher than that in normal control group 3 weeks after injection with Iso. The expression level of CTGF increased with the increasing severity of MF. However, after intervention with SF, both the transcription level of CTGF mRNA and the expression level of CTGF decreased significantly. Conclusion The expression of CTGF was closely related to the severity of MF of rats, indicating the potentially important role of CTGF in MF. However, SF showed inhibitory effect on expression of CTGF.

    2010 08 v.23 [Abstract][OnlineView][Download 393K]

  • Comparison of Determination Results by HIV Antibody Confirmation Kits Manufactured by Various Manufacturers

    ZHANG Chun-tao, LI Xiu-hua, SONG Ai-jing, XU Si-hong, NIE Jian-hui, WANG You-chun(National Institute for the Control of Pharmaceutical and Biological Products, Beijing 100050, China)

    Objective To compare the determination results by HIV antibody confirmation kits manufactured by three manufacturers. Methods Sixty-four specimens with inconsistent results by several ELISA kits for HIV antibody were confirmed by the HIV antibody confirmation kits manufactured by three manufacturers, and the results were compared, based on which the common nonspecific bands in uncertain specimens were analyzed. Results All the 64 specimens were confirmed as negative or uncertain by the HIV antibody confirmation kits manufactured by three manufacturers. However, the proportions of negative and uncertain specimens confirmed by various kits were significantly different. The coincidence rates of confirmation results by INNO-LIATM HIVⅠ / Ⅱ Score and NEW LAV BLOT 1 kits, INNO-LIATMⅠ / Ⅱ Score and HIV BLOT 2. 2 kits, and NEW LAV BLOT 1 and HIV BLOT 2. 2 kits were 57. 8%, 28. 1 and 48. 4%, respectively. Significant differences were observed between the confirmation results by various kits(P < 0. 01). Uncertain results were mainly caused by p24 and p17 bands. Conclusion The qualities of HIV antibody confirmation kits are significantly different, indicating that it is necessary to strengthen the quality control and regulate the requirements for the kits.

    2010 08 v.23 [Abstract][OnlineView][Download 117K]

  • Safety and Immunogenicity of Groups A + C Meningococcal Conjugate Vaccine

    SHI Nian-min, QU Yan, AI Xing, PANG Run-tian, BAI Yun-hua, YANG Li-qing, WU Jiang (Center for Disease Prevention and Control of Chaoyang District, Beijing, Beijing 100021, China)

    Objective To observe the safety and immunogenicity of freeze-dried groups A + C meningococcal conjugate vaccine. Methods A double-blind, randomized and parallel controlled clinical trial was carried out in the children at ages of 6 ～ 9 months and not less than 3 years. The children in trial groups were immunized with freeze-dried groups A + C meningococcal conjugate vaccine. However, in control groups, the children at ages of 6 ～ 9 months were immunized with group A meningococcal polysaccharide vaccine, while those at ages of not less than 3 years with groups A + C meningococcal polysaccharide vaccine. The adverse reaction, serum antibody content and antibody positive conversion rate after immunization were observed. Results The systemic adverse reaction rates in the children at ages of 6 ～ 9 months in trial and control groups were 1. 67% and 4. 58%, while those of local adverse reaction rates were 0. 83% and 2. 08%, respectively. However, the systemic adverse reaction rates in the children at ages of not less than 3 years in trial and control groups were 2. 50% and 3. 33% respectively, while those of local adverse reaction rates were both 0. 83%. The contents of antibody against group A meningococcus in sera of children at ages of 6 ～ 9 months in trial and control groups were 22. 27 and 3. 61 μg / ml, while the antibody positive conversion rates were 99. 01% and 66. 67%, respectively, which showed significant difference(P < 0. 05). The contents of antibodies against groups A and C menigococcus in sera of children at ages of not less than 3 years in trial group were 21. 28 and 17. 94 μg / ml, while the antibody positive conversion rates were 99. 04% and 98. 08%, respectively. However, in the sera of children at ages of not less than 3 years in control group, the contents of antibodies against groups A and C menigococcus were 34. 43 and 22. 66 μg / ml respectively, while the antibody positive conversion rates were both 98. 04%. The antibody contents of children at ages of not less than 3 years in trial and control groups showed significant difference(P < 0. 05), while the antibody positive conversion rates showed no significant difference(P > 0. 05). Conclusion Freeze-dried groups A + C meningococcal conjugate vaccine showed high safety and immunogenicity in the children at ages of 6 ～ 9 months and not less than 3 years.

    2010 08 v.23 [Abstract][OnlineView][Download 152K]

  • Genotyping and Genetic Background of A Rh D-- Individual and Her Family Members

    WU Wei-jian, GUO Ru-hua, YU Jin-lin(Foshan Blood Center, Foshan 528000, Guangdong Province, China)

    Objective To analyze the genotype and genetic background of a Rh D--individual and her family members. Methods The specific sequences of RHD and RHCE genes of a Rh D--individual and her family members were amplified by PCRSSP and analyzed. Results The PCR results of family members of the index case were basically consistent with their serological phenotypes. The RHD gene of index case showed no abnormality, while no specific sequence of RHCE gene was detected. However, the phenotypes and PCR products of RHCE genes of her family members were normal. Conclusion The important molecular basis of Rh D--was partial or complete deletion of RHCE gene of index case, which was deduced to be due to the two D--haplotypes carried by her father and mother respectively.

    2010 08 v.23 [Abstract][OnlineView][Download 386K]

  • Advance in Research on Human CD4 Molecule and Anti-CD4 Therapeutic Antibodies

    HU Di-chao, ZHANG Ai-hua, YANG Xiao-ming(Wuhan Institute of Biological Products, Wuhan 430060, China)

    Normally, human CD4 molecule can aid the immune system with immune defense, immune homeostasis and immune surveillance. Under some abnormal circumstance, however, it closely correlates with the occurrence and progression of some diseases. At present, several therapeutic antibodies against CD4 molecule have been developed, and certain achievements have been made in clinical study on some diseases. This paper reviews the advances in research on the structure and function of human CD4 molecule, the relationship between CD4 molecule and some diseases as well as anti-CD4 therapeutic antibodies.

    2010 08 v.23 [Abstract][OnlineView][Download 174K]

  • Progress in Research on Targeted Anti-HIV Therapeutic Agents

    HAN Jia-li, LI Chang, JIN Ning-yi (The Military Veterinary Institute, Academy of Military Medical Sciences, Changchun 130062, China)

    The principle of anti-HIV therapy is selective delivery of bioactive warheads killing the target cells of HIV infection to the infected target cells by a specific vehicle, which is internalized after binding to the target cells then kill the residual virus particles and latently infected cells, repair the immune system and recover the normal function of organism. As an accessory and supplemental therapy, targeted anti-HIV therapy is becoming is novel method for treatment of AIDS. The progresses in research on vehicle and bioactive warheads for targeted anti-HIV therapy are reviewed in this paper.

    2010 08 v.23 [Abstract][OnlineView][Download 167K]

  • New Strategy of Blood Screening for Hepatitis B Virus

    BAI Yu, CHANG Wei-hong (Center for Drug Evaluation, State Food and Drug Administration, Beijing 100038, China)

    There is a high prevalence of hepatitis B virus (HBV)in China where the prevention and control of transfusiontransmitted hepatitis B has been paid more and more attention. The introduction of novel techniques, such as nucleic acid testing (NAT), is one of the important measures to decrease the risk of HBV infection and increase the blood safety. In this paper, the status of application of NAT for HBV in blood screening is reviewed, and the strategy of blood screening for HBV is investigated.

    2010 08 v.23 [Abstract][OnlineView][Download 137K]

  • Progress in Research on Antibacterial, Antiviral and Immunopotentiation Effects of Tea Components

    FANG Chong-ye, FU Xue-qi, SHENG Jun(College of Life Science, Jilin University, Changchun 130061, China)

    Tea drinking is of many benefits to the health of humans, such as antioxidation, anti-aging, antitumor and decreasing the morbidities of some diseases. In recent years, the antibacterial and antiviral effects as well as immunopotentiation of tea components have been reported increasingly. The progress in research on the antibacterial, antiviral and immunopotentiation effects of major tea components, such as tea polyphenol, tea pigment, tea polysaccharide and theanine, are reviewed in this paper.

    2010 08 v.23 [Abstract][OnlineView][Download 108K]
    • 下载本期数据

@2012 Editorial Department of "Chinese Journal of Biologicals"