【作者】 丁生财；

【导师】 陈意生； 魏泓；

【作者基本信息】 第三军医大学 ， 病理学与病理生理学， 2002， 博士

【摘要】 猪器官、组织和细胞作为临床移植的供体源具有广泛和良好的前景，可缓解临床器官移植的器官相对短缺，也可为临床治疗Ⅰ-型胰岛素依赖型糖尿病，帕金森病、亨廷顿病、癫痫病等顽固性神经疾患提供一种治疗方法，并可作为治疗急性爆发性肝炎肝功能衰竭、急、慢性肾功能衰竭的过渡性措施。自Dr．Clive Patience(BioTransplant Inc．首席科学家)1997年报道猪内源性逆转录病毒(PERV)感染体外培养人细胞以来，猪异种移植过程中可能发生PERV种间传播的潜在危险已引起众多学者的广泛关注。 Patience等首先证实至少有三个PERV亚型在体外培养能感染人细胞，Takeuchi等通过假型实验也证实PERV感染体外培养人细胞株，Martin等通过感染性实验证实了PERV感染体外培养人细胞。以后许多学者从多方面对PERV的生物学特性进行了较为广泛和深入的研究。 目前已经证实猪内源性逆转录病毒为典型的哺乳动物C型逆转录病毒，并具有gag-pol-env基因组结构。Patience等从两株猪肾细胞株分离得到的天然病毒粒子，证实为典型的哺乳类C型逆转录病毒。Akiyoshi等鉴定小型猪淋巴细胞PERV(PERV-MSL)全长cDNA核酸序列，证实PERV-MSL是一种典型的C型逆转录病毒，其长度为8132bp，与长臂猿白血病病毒和鼠白血病病毒具有最大的核酸序列同源性。同时在多种猪器官和正常白细胞检出PERV-MSL RNA的结构产物。PERV-MSL的gag、pol、env的ORF氨基酸序列与Tsukuba-1逆转录病毒氨基酸序列的同源性＞99％，与胎猪肾细胞PERV(PK15-PERV)氨基酸序列具有高度同源性。我国猪种资源丰富，国内尚未开展猪内源性逆转录病毒生物学方面的实验研究。 体外实验已经证实了猪PERV对人细胞具有感染性，但在体感染性研究报道甚少。Van der Laan等通过建立非肥胖性糖尿病／严重联合免疫缺陷wOD用CID鼠胰岛细胞移植模型，首先证实猪胰岛细胞产生PERV，并在体外培养感染人细胞，并移植至NOD侣 鼠，检测鼠组织PEM RNA的表达，结果发现多种组织发生PERV感染，这是移植猪组织后体内PERV主动复制，也是在体种间交叉感染的首次报道。后来Deng等建立SCID鼠移植胎猪胰岛细胞模型，将胎猪胰岛移植于 SCID鼠，饲养 3＊ 周分别处死。取肝、脾、脑提取 DNA和 RNA，应用巢式 PCR和 RTPCR检查 PEM和扩增猪线粒体细胞色素氧化酶亚单位＊基因o），结果发现M4－肝和M19－脾组织的PERV阳性，COil阴性，这提示猪PERV向鼠组织传播己经发生，但检测 PERV RNA未发现鼠组织有活跃的 PERV复制的证据。这些结果初步表明猪细胞可以在体感染鼠组织。而近期有几宗接受活猪组织或细胞移植的临床病例的文献报道，但所有报道均表明未发现PERV感染的证据。临床结果与实验结果并不完全一致，因此PERV的在体感染性尚末到广泛的公认，有待进一步深入研究。 为此，本课题拟从猪外周血白细胞基因组存在的PERV基因序列及其差异性、各种猪组织PERV基因序列的mRNA丰度、猪皮肤成纤维细胞对人HEK293细胞的体外感染性和对SCID鼠的在体感染性入手，研究我国猪PERV的生物学特性、猪组织PERV的表达的高低幅度（高表达和低表达）及猪皮肤成纤维细胞PERV的体外和体内感染性，从而为猪－人间异种移植积累资料，为临床应用提供实验依据，为异种移植的生物安全性评价奠定基础。研究内容分为以下三部分：＊）猪外周血白细胞DNA猪内源性逆转录病毒序列的检测及差异性分析。主要了解我国猪资源的PERV生物学特性，为猪异种移植积累资料。p）两种小型猪不同组织 PERV m洲A丰度及其差异分析。了解备种组织PERV表达情况，以寻找PERV高表达和低表达器官或组织，为临床猪异种移植选择组织或器官提供实验依据。O）猪皮肤成纤维细胞PERV的体外和体内感染性实验研究。通过实验了解猪皮肤成纤维细胞PERV的对人HEK293细胞的体外感染性和对SCID鼠的体内感染性，为猪异种移植提供实验依据。 ·x· 本研究应用PCR、PCR－RFLP、SS－SSCP等技术分析12个品系猪外周 血白细胞基因组DNA的PERV基因序列片段及其差异，应用RT－PCR方 法分析广西巴马小型香猪和贵州小型香猪13种组织P＊RV基因序列m RNRNA 的表达及其差异，应用组织细胞培养方法、免疫组织化学、电镜技术建立 和鉴定猪皮肤成纤维细胞系，并将猪皮肤成纤维细胞与经过转染的具有抗 新霉素基因的人neo／HEK293细胞共培养，或将猪皮肤成纤维细胞移植于 SCID 鼠，探讨猪内源性逆转录病毒的体外和体内感染性，获得以下结果 和结论： 1．口个品系猪外周血白细胞 DNA池基因组普遍存在 PERV－A、 PERVI、PERV gag基因序列，分析各品系个体发现PERV（基因序列的 检出率约为 10％。应用逆转录病毒逆转录酶基因高度保守区序列为引物， 寻找新的逆转录病毒序列，未能发现新的逆转录病毒序列；应用SS七SCP 技术分析 12个品系猪外周血白细胞基因组扩增产?更多还原

【Abstract】 It is of broad and charming feature that organs, tissues and cells of the pig as donor source for xenotransplantation to human beings. It may alleviate the relative shortage of human donor organs, and provide not only a substitutive therapy for patients suffering from intractable neurologic disorders (Parkinson’s disease, Huntington’s disease, and epilepsy) and type- I insulin-dependent diabetes mellitus, but also a interim measure for patients suffering from acute fulminant hepatitis with liver failure and acute and chronic renal failure. Since Dr. dive Patience (a principal scientist at BioTransplant Inc.) reported firstly porcine endogenous retrovirus (PERV) might infect human cells in vitro in 1997, extensive attentions have been attracted on the potential hazard of interspecies infection with xenotransplantation to human beings.Patience et al has demonstrated three variants of PERV that could infect human cell in vitro at least. Takeuchi et al has shown that PERV could infect human cell lines in vitro by pseudotyping experiments. Martin et al has confirmed PERV could infect human cell in vitro by infectious trial. More extensive and deeper researches about biological characteristics of PERV have been performed by many of scholars.It has been demonstrated that PERV is a typical mammalian C-type retrovirus with a comlpex structure of gag-pol-env gene. Patience et al seperated natural virus particles from two pig kidney cell lines, and’The project is supported by the national KEY basic research developmental programe. No.G2000016106demonstrated a typical mammalian C-type retrovirus particles. Akiyoshi et al reported the nucleotide sequence of porcine endogenous retrovirus from lymphocytes of miniature swine (PERV-MSL) is a typical mammalian C-type retrovirus of 8,132 bp with the greatest homological nucleic acid sequence to gibbon ape leukemia virus and murine leukemia virus. Constitutive product of PERV-MSL RNA has been detected in normal leukocytes and multiple organs of swine. The open reading frames for gag, pol, and env in PERV-MSL have over 99% homological amino acid sequence identity to those of Tsukuba-1 retrovirus and are highly homologous to those of endogenous retrovirus of cell line PK15 (PK15-ERV). Sources of Pig species are rich in China. Experimental study of biological features of PERV has not been reported domestically so far as we know.Although PERV can infect human cell in vitro has been demonstrated, few research that PERV is transcriptionally active and infectious cross-species in vivo was reported. Van der Laan et al established a model of pig pancreatic islets transplanted into NOD/SCID (non-obese diabetic mellitus, severe combined immunodeficiency) mice. Here they showed that cells of pig pancreatic islets produced PERV and infected human cells in culture. After transplantation into NOD/SCID mice, the expression of PERV RNA in mice was detected. The results showed that various tissues of mice were infected by PERV. This is the first report that PERV is active replication and infectious interspecies in vivo after transplantation of pig tissues. Subsequently, Deng et al transplanted twenty-one SCID mice with fetal pig pancreatic cells and brought up for three to 41 weeks before being killed. DNA and RNA were extracted from the liver, spleen, and brain of these mice, and examined for PERV using nested polymerase chain reaction (PCR) and reverse transcriptase-PCR. The pig mitochondrial cytochrome oxidase II subunit gene (COII) was also amplified to monitor the presence of pig cell microchimerism in xenotransplanted tissues. The results showed the PERV was positive inM4-liver and Ml9- spleen, and COII negative. This indicated that transmission of PERV from pig to mouse had happened. The negative reverse transcriptase-PCR results for PERV from these mouse samples suggest there was no active PERV replication in the mouse tissues. No evidence of infection with PERV was found recently in a several of reports on clinical patients transplanted alive tissues or cell of更多还原