摘要
目的通过胶体金探针标记乙肝病毒(HBV)全基因转染的人胎盘滋养细胞(HPT-8-HBV),示踪乙肝表面抗原(HBsAg)在HPT-8-HBV细胞中的分布。方法通过脂质体介导的方法将HBV全基因转染HPT-8细胞,G418筛选,通过ELISA、免疫组化对转染细胞进行检测;采用胶体金标记的HBsAg单克隆抗体作为探针(简称胶体金探针)对转染细胞中的HBsAg进行标记示踪。结果成功将质粒pcDNA3-3HBV转染到HPT-8,转染后第1、2代培养上清ELISA检测显示HBsAg阳性,免疫组化检测第2代转染细胞中HBsAg阳性;通过胶体金探针标记发现HBsAg存在于转染滋养细胞中的粗面内质网、分泌泡、空泡和溶酶体内,以及细胞膜和绒毛上。结论可以从重组质粒pcDNA3-3HBV转染的滋养细胞的树脂切片中获得HBsAg的超微结构定位。
Objective To trace the distribution of hepatitis B surface antigen （HBsAg） in human placental trophoblast （HPT-8-HBV） cells transfected by pcDNA3-3HBV using anti-HBs bound colloidalGold probes. Methods HPT-8 cells were transfected with peDNA3-3HBV and screened with antibiotic G418. The transfected cells were detected by ELISA and irrrmunocytoehemical staining. HBsAg in transfected ceils were marked and traced using an anti-HBs bound colloidal gold probes, combined with transmission electron microscopy technique. Results The established trophoblast cells were then stably transfected with pcDNA3-3HBV. The HPT-8-HBV also produced and secreted HBsAg at passage 1 and 2. Immunocytochemistry method detected HBsAg in HPT-8-HBV cells at passage 2. The HBsAg was visualized at the ultrastructural level in HPT-8-HBV cells by immune-transmission electron microscope techniques using colloidal gold particles as labels. Ultrastruetural study confirmed that colloidal gold particles bind specifically to HPT-8-HBV cells villus, membrane, cytolysosome, rough endoplasmic reticulum, secretory vacuole and vacuole. Conclusions Ultrastruetural localization of HBsAg can be obtained in sections of HPT-8-HBV cells resin.
出处
《中华疾病控制杂志》
CAS
2010年第2期99-103,共5页
Chinese Journal of Disease Control & Prevention
基金
国家自然科学基金重点课题(30230320)
陕西省自然科学基金(2006C248)
关键词
肝炎表面抗原
乙型
滋养细胞
探针
转染
Hepatitis B surface antigen
Trophoblast cell
Probe
Transfeetion
