猪内源性逆转录病毒体外感染人胚肾细胞系HEK-293的初步研究

Preliminary study on human embryonic kidney cell line HEK-293 after porcine endogenous retrovirus infection

目的 对猪内源性逆转录病毒(PERV)体外感染人胚肾细胞系HEK 293的能力进行检测。方法 建立体外感染人胚肾细胞系HEK -293的方法,用免疫电镜的方法观察PERV粒子,聚合酶链反应(PCR)、逆转录聚合酶链反应(RT- PCR)检测与PERV共培养24h后HEK 293细胞基因组中PERV前病毒基因的整合、表达并对PERV亚型进行鉴定,激光共聚焦显微镜检测PERV -gag蛋白的表达并进行定量分析。结果 电镜下可观察到PERV的圆形病毒粒子;与PERV共培养24h后HEK -293细胞基因组中检测到PERV前病毒DNA,且mRNA也有效表达,PERV亚型为PERV -A,B型;激光共聚焦显微镜观察到PERV -gag蛋白在感染细胞HEK- 293的胞质表达,但表达量下降。结论PERV具有体外感染人胚肾细胞系HEK- 293的能力,病毒蛋白可以有效表达,因此在猪到人的异种器官移植研究中对PERV可能引起的人兽共患病进行安全性评价是极有必要的。

Objective To assess the infectivity of porcine endogenous retrovirus (PERV) via in vitro infection of human embryonic kidney cell line HEK-293. Methods PERV particles were detected by immunoelectron microscopy. PERV DNA a nd mRNA were studied in HEK-293 24 hours after the infection using polymerase c hain reaction and reverse transcriptase-PCR respectively. The PERV types were a lso analyzed. PERV-gag protein was observed by confocal microscopy. Results Retroviral particles were round under electron microscope. PERV-gag pol gene and gag protein were detected and expressed in the infected HEK-293 ce lls. The types of PERV were PERV-A and PERV-B. PERV-gag protein was also iden tified in the cytoplasm of infected cells by confocal microscopy. Conclusions PERV is able to infect HEK-293 cell line in vitro; types of PERV -gag protein is also expressed as a result. Further studies are thus necessary in order to evaluate the possibility of xenozoonoses in pig-to-human xenotrans plantation.