RT-SHIV中国恒河猴适应株细胞水平生物学特性分析  

In Vitro Characterization of RT-SHIV Adapted in Chinese Rhesus Monkeys

作　　者：姚南[1] 王卫[1] 丛喆[1] 金光[1] 陶真[1] 魏强[1] 

机构地区：[1]中国医学科学院医学实验动物研究所卫生部人类疾病比较医学重点实验室国家中医药管理局人类疾病动物模型三级实验室,北京100021

出　　处：《中国比较医学杂志》2011年第2期31-35,79,共6页Chinese Journal of Comparative Medicine

基　　金："十一五"国家科技重大专项课题(2009ZX10004-307);协和青年基金(RT-SHIV动物模型的建立)

摘　　要：目的体外增值、制备动物感染来源的RT-SHIV病毒中国恒河猴适应株,比较PBMCs和CEMx174两种细胞制备出病毒的差异,同时用TZM-bl、CEMx174、PBMC三种细胞滴定测定病毒TCID50。方法用RT-SHIV病毒静脉感染中国恒河猴,定期采血测定血浆病毒载量,当病毒载量达高峰时采血分离外周血单核淋巴细胞(PBMCs),与正常恒河猴PBMCs或CEMx174细胞共培养,定期测定培养液中的P24抗原水平,当病毒复制达高峰期时收集培养上清,分装并冻存;测定病毒RNA载量、P24抗原浓度,滴定病毒的TCID50。结果本研究共制备了78 mL PBMCs来源的RT-SHIV病毒和85 mL CEMx174细胞来源的RT-SHIV病毒。RT基因序列和原始序列的相似度为99%,仅在第254和265位的氨基酸发现突变。RT-SHIV(PBMC)和RT-SHIV(CEMx174)病毒载量分别为1.641×108 copies/mL和8.375×108 copies/mL,P24抗原水平分别为20.745 ng/mL和4.28 ng/mL,TZM-bl、CEMx174、PBMC细胞测定病毒的TCID50分别为3.16×105 TCID50/mL和1×104 TCID50/mL,5×102 TCID50/mL和5×105 TCID50/mL,5×102 TCID50/mL和5×103 TCID50/mL。结论 PBMCs细胞来源制备的病毒较CEMx174制备的病毒具有更高的感染性。Objective To propagate and titrate the adapted RT-SHIV from Chinese rhesus monkey using TZM-bl,CEMx174 and PBMCs cells,and to compare the possible difference of these viruses which propagated in PBMC and CEMx174,respectively.Methods Rhesus macaques free-living in China were infected with RT-SHIV intravenously.Blood samples were collected regularly and viral loads were monitored.Peripheral blood mononuclear cells（PBMCs） were examined when the viral load reached the peak,and were co-cultured with PBMCs from uninfected rhesus or CEMx174 cells.The P24 antigen level from supernatant was monitored regularly.The co-cultured virus was collected,packed and frozen when the viral replication reaches the peak.The collected viruses as a stock were charactarized with the RNA viral loads,P24 antigen level and titration of TCID50.Results In this study,in tatol of 78 mL of RT-SHIV in monkeys’ PBMCs and 85 mL of RT-SHIV was propagated in CEMx174 cells,respectively.The similarity between RT gene sequence and original sequence was 99%,only two mutation of amino acid at 254 and 265.RT-SHIV（PBMC） and RT-SHIV （CEMx174）viral loads were titrated with TZM-bl,CEMx174 and PBMCs,and their results were 1.641 × 108 copies/mL and 8.375 × 108 copies/mL.respectively,P24 antigenlevel were 20.745 ng/mL and 4.28 ng/mL,respectively,titrations of TCID50 in TZM-bl,CEMx174,PBMC were 3.16 × 105 TCID50/mL and 1 × 104 TCID50/mL;5 × 102 TCID50/mL and 5 ×105 TCID50 /mL;5 × 102 TCID50/mL and 5 × 103 TCID50/mL,respectively.Conclusions RT-SHIV propagated from PBMCs has higher infective ablity than that from the other cells in vitro.

关 键 词：RT-SHIV TCID50 细胞培养 

分 类 号：R373.33[医药卫生—病原生物学] R33[医药卫生—基础医学]