原代人绒毛滋养层细胞分离培养及其对巨细胞病毒的易感性研究

Primary Culture of Human Trophoblastic Cells and Their Susceptibility to Huma Cytomegalovirus

目的通过分离培养原代人绒毛滋养层细胞(HTCs)并检测其对人巨细胞病毒(HCMV)的易感性,探讨胎儿宫内感染HCMV的可能途径。方法将30例健康孕妇经负压真空吸引手术所得的流产早孕绒毛组织充分剪碎,用0.25%胰蛋白酶和0.2%DNA酶混合消化法制备细胞悬液,细胞悬液经200目不锈钢筛网过滤后用Percoll液密度梯度离心对HTCs进行初步纯化,细胞传代中应用反复胰酶消化法对HTC进行3～5次纯化,通过细胞学形态观察和免疫荧光染色方法对HTCs进行鉴定;用HCMV病毒株体外感染HTCs,应用PCR、Westernblot和免疫荧光方法对HCMV基因及蛋白进行检测,从而确定HTCs对HCMV的易感性。结果 Percoll分离联合胰酶、DNA酶消化法对原代HTCs有较好的分离效果,免疫荧光染色结果表明90%以上细胞表达CK-7相关抗原,HTCs体外感染HCMV 72h后形态学发生明显改变,病毒基因及蛋白均呈阳性表达。结论原代HTCs对HCMV易感,通过HTCs感染胎儿可能是HCMV宫内感染的重要传播途径之一。

Objective to determine the susceptibility of primary human trophoblastic cells（HTCs） to human cytomegalovirus（HCMV） infection.Methods 30 villus of healthy pregnant women were digested with a mixture of 0.25% trypsin and 0.2% DNase.The digestion was then filtered by a 200 mesh stainless steel sieve.HTCs were purified by Percoll gradient centrifugation and repeat purification with trypsin during cell passage.HTCs were identified by cell morphology and immunofluorescence.PCR,Western blot and immunofluorescent staining were performed to detect HCMV gene and protein 72 hours post infection of HCMV.Results The combination of 0.25% trypsin and 0.2% DNase with Percoll gradient centrifugation isolated and purified primary HTCs.More than 90 percent cells expressed CK-7 related antigen.The results of PCR,western blot and immunofluorescence showed positive expressions of CMV gB and virus proteins.Conclusion HTCs is susceptible to HCMV infection,which suggests a possible route of HCMV infection at the fetal-maternal interface.