【作者】 陆彩霞；

【导师】 代解杰； 孙晓梅； 高家红；

【作者基本信息】 中国协和医科大学 ， 动物学， 2009， 硕士

【摘要】 SHIV病毒感染艾滋病动物模型是目前国外艾滋病模型研究的趋势,被认为是比现阶段普遍应用的SIV模型针对性更强、更有意义的动物模型。国内也已开始SHIV病毒感染艾滋病动物模型的研究,本研究以SHIV-KB9(以SIVmac239病毒基因组和HIV B亚型env、tat、rev和vpu基因为主体构建的一株SHIV病毒)感染已导入MazF基因和未导入MazF基因的中国猕猴,进行了包括临床表现、病毒血症、免疫学和基因拷贝数等方面研究。本研究共选用了9只中国猕猴,实验前经血清学间接免疫荧光抗体检查法(IFA)检查排除猴免疫缺陷病毒(SIV)、猴T淋巴细胞性I型病毒(STLV-1)和猴逆转录D型病毒(SRV-1、2、5)的感染和B病毒感染。分三组进行,空白对照组1只(只进行SHIV-KB9病毒攻击),编号T-bc,实验对照组2只(未导入基因),编号KUCD4+T01(T01)和KUCD4+T02(T02);实验组6只(导入基因),编号T-Mar、T-Jun、KUCD4+T03(T03)、KUCD4+T04(T04)、KUCD4+T05(T05)和KUCD4+T06(T06),T-Mar和T-Jun动物进行了基因导入未用SHIV-KB9病毒攻击。采用体外循环采集猕猴外周血PBMC和直接抽取猕猴外周血75mL分离PBMC,再进行CD4+T细胞分离培养并导入基因,培养结束后收获细胞静脉回输猴体后再进行SHIV-KB9病毒感染。分别在感染前和感染后第0、3、8、11、14、17、21、24、28、35、49、56、63、70、84、98天用EDTA抗凝管采集猴静脉血3mL,取100ul抗凝血进行血常规分析,其余抗凝血离心分离血浆用于病毒载量检测,以监测SHIV-KB9在猴体内的复制;分离PBMC用于CD4~+/CD8+值测定,以监测SHIV-KB9感染实验猴后体内免疫损伤情况。实验结果显示导入基因和回输细胞的实验猕猴其血浆病毒载量峰值都与已报道的推迟2周左右出现,比空白对照组推迟11天出现,其血浆病毒载量峰值比空白对照组峰值低约100倍,流式结果表明其CD4+T细胞计数也稳定在一定水平,CD4/CD8比值未出现明显倒置。实验组猕猴在试验结束时均未发展为猴AIDS。结论:导入CD4+T细胞的MzaF基因在治疗猕猴AIDS模型中显示了一定的效果,能延缓血浆病毒载量出现的时间,但更确定的效果还有待进一步的研究。更多还原

【Abstract】 SHFV infection of rhesus macaques provides a reliable and popular model to study HIV/ADDS on abroad, and it is better than SFV infection animal models. SHIV animal models are now undertook in our country. In this research, rhesus macaques of Chinese origin, which were transduced MazF gene or not transduced, were inoculated intravenously with 10 MID50 of SHIV-KB9, we monitored clinical manifestation, viremia, immunology and gene copies.9 rhesus macaques were used in this study. All the rhesus macaques were screened by serological test to ensure that they were free of SFV, SRV , STLV and BV infection.They were divided into three groups, blank group(T-bc), control group(T01,T02) and experimental group(T03,T04,T05,T06,T-Mar and T-Jun). PBMC were collected by Cobe Spectra blood cell separators and by drawing 75mL blood, then to isolate CD4+T and proliferate ex vivo and transduce MazF gene. When cell culture was over, to harvest the cells(transduced or not transduced) and re-infuse the cells into rhesus macaques. Finally, all rhesus macaques were inoculated with SHTV-KB9,except T-Mar、T-Jun and T-bc. Blood samples were collected at different time according to the time schedule, to do blood composition anaysis, and prepare both plasma and PBMC for viral RNA by Real Time RT-PCR and FACS / MazF gene copy assay, respectively.The results showed the viral load peak was put off 11 days contrast to blank group and about 2 weeks late according to documents, the level of plasma viremia was lower about 100 times than blank group, CD4+T cell count and CD4/CD8 ratio were stable. All animals did not involved in AIDS at the end of this experiment.Conclusion: Re-infusing the transduced cells into monkeys can delay the viral load peak, it is partially useful in therapy SHIV/AIDS animal models, but the exact effect of MazF will be reseached further.更多还原