HBsAb标记胶体金探针对HBV阳性血清感染HPT-8细胞的示踪研究

Tracer Study on HBV Positive Serum Infection of Human Trophoblast Cells(HPT-8) with Anti-HBs Bound Colloidal Gold Probes

目的：为临床诊断提供HBV进入HPT-8的依据。方法：HBV阳性血清（1．5×10^8拷贝／毫升）体外感染HPT-8细胞48h，设感染组、阴性对照组和空白对照组，ELISA检测细胞培养上清中HBsAg、HBeAg，提取感染后传代的各代细胞中DNA用于PCR检测HBVDNA。免疫荧光定量PCR检测细胞培养上清中HBVDNA的滴度。采用乙肝表面抗原单克隆抗体标记的胶体金探针对感染的HPT-8细胞中的乙肝表面抗原进行标记示踪。结果：ELISA检测感染组细胞第1、2代培养上清中HBsAg阳性，PCR检测感染组第1、2代细胞HBVDNA，结果均阳性。免疫荧光定量PCR检测感染组细胞上清在第1、2代、3代48h的HBV滴度分别为8×10^4、4．6×10^3〈10^3（拷贝／毫升）。通过胶体金探针的标记：乙肝表面抗原存在于感染细胞的溶酶体内、细胞膜和绒毛上。结论：血清中的HBV病毒能进入胎盘滋养细胞，并可从感染滋养细胞的树脂切片中获得乙肝表面抗原的超微结构定位。

Objective： To provide the evidence of HBV positive serum infecting human trophoblast cells （HPT-8） by molecular biology technique. Methods： Diluted HBV positive serum （ 1.5 × 10^8 copy/ml） was used to infect HPT8 cells for 48h. Infection group, negative control group and blank control group were studied. ELISA method was used to detect HBsAg and HBeAg in cell culture medium. PCR method was used to detect HBV DNA in every passage of HBV infected cells. Real-time fluorescence quantitative PCR was used to detect the titer ofHBV DNA in cell culture medium. HBsAg in HBV infected cells were examined by using anti-HBs bound（HBsAb） colloidal gold probes, combined with transmission electron microscopy technique. Results： HBsAg was positive in cell culture medium in Passage 1 and 2 of infected group detected by ELISA method. HBV DNA was positive in cell culture medium in Passage 1 and 2 of infected group detected by by PCR. In cell culture medium in Passage 1 ,2 and 3 of infected group detected by fluorescence quantitative PCR, the titers of HBV DNA for 48h were 8 × 10^4,4.6 ×10^3,〈 10^3（copy/ml） respectively. Conclusion： Through HBsAb marker, it was confirmed that HBsAg existed in the lysosomes, cell membranes and villi of infected cells. HBV positive serum could infect HPT-8 cells in vitro. Specific labeling by colloidal gold probes could obtain the ultrastructural localization of HbsAg from the resin sections of infected HPT-8 cells.