HBV感染者胎盘组织HBV ccc DNA检测  

Detection of HBV ccc DNA in placental tissue of HBV carrier

作　　者：陈京龙[1] 刘霞[2] 谢尧[3] 兰孟东[4] 

机构地区：[1]首都医科大学北京地坛医院肿瘤内科,北京100015 [2]山东大学齐鲁儿童医院内一科,济南250013 [3]首都医科大学北京地坛医院内4科,北京100015 [4]首都医科大学北京地坛医院病理科,北京100015

出　　处：《中国肝脏病杂志（电子版）》2009年第1期25-30,共6页Chinese Journal of Liver Diseases：Electronic Version

基　　金：首都医学发展基金项目(编号:2002-2016)

摘　　要：目的建立HBV ccc DNA在胎盘组织中定性及定位检测方法,了解慢性HBV感染者胎盘组织中HBV ccc DNA的存在状况,进一步探讨其意义及乙型肝炎病毒母婴传播机制。方法选取首都医科大学北京地坛医院慢性乙型肝炎病毒感染者孕晚期胎盘组织40例,阳性对照肝组织标本3例,阴性对照为健康足月妊娠产妇胎盘标本,共5例。所有胎盘标本均分两份,分别于-80℃冷冻和福尔马林室温浸泡保存。冷冻标本采用质粒抽提法提取胎盘组织中HBV ccc DNA,液相PCR定性检测;福尔马林处理标本作石蜡切片,用于原位PCR定位检测。结果 40例标本中,液相PCR检测出HBV ccc DNA阳性者8例,其相应的血清HBV DNA均>105拷贝/ml。取该8例及另2例乙型肝炎患者孕晚期胎盘石蜡切片经HBV ccc DNA原位扩增,7例检测出HBV ccc DNA阳性,胎盘绒毛毛细血管内皮细胞、绒毛合体滋养层细胞、PBMC等多种细胞均存在HBV ccc DNA阳性信号。结论研究表明,胎盘组织HBV感染与孕妇血液中HBV DNA含量显著相关。胎盘组织的HBV ccc DNA原位PCR检测显示,HBV可感染胎盘各层细胞。羊膜上皮细胞中也发现HBV ccc DNA阳性信号。胎盘组织感染的HBV及其在这些细胞中的复制是否直接与HBV宫内感染相关尚需进一步研究。Objective To construct the methods of HBV ccc DNA qualitation and localization in placental tissue and study the HBV ccc DNA status in placent of HBV carrier, furtherly to approach the significance of detection and the mechanism of intrauterine transmission. Methods Forty cases of HBV carrier placental tissue of late trimester of pregnancy in Beijing Ditan Hospital, twenty cases with serum HBV DNA > 105 copies/ml, and the other 20 cases with serum HBV DNA < 105 copies/ml. Three positive hepatic tissue and 5 placent of pregnancy at term were enrolled as positive control and negative control, respectively. All samples were divided into two groups, stored in -80℃ and soaked in formaldehyde at room temperature, respectively. HBV ccc DNA was extracted from freezed tissue through plasmid extraction and stored for PCR detection; paraffin section was carried out for isPCR. Results Liquid phase PCR indicated that 8 cases of 40 placental samples were HBV ccc DNA positive, whose serum HBV DNA > 105 copies/ml. We qualitatively considered that hepatitis B virus reproduced in the placent tissue. Placent paraffin section in situ amplification was carried out for 10 cases, 7 cases were positive with royal blue signal and located in the cell nucleus of placental villus syncytial trophoblas, capillary endothelium, pile interstitial substance, PBMC interior blood capillary and amniosis cellula epithelialis. By comparing HE dyeing with alkali phosphatase dyeing,we confirm that HBV ccc DNA is present in parts of vascular endothelial cell, PBMC and so on. Conclusions HBV ccc DNA was detected in capillary endothelium of chorion, capillary endothelium and PBMC. Liquid phase PCR indicate that 8 cases of 20 placental samples with serum HBV DNA > 105 copies/ml which were HBV ccc DNA positive, intrauterine transmission is related to high HBV DNA content. Seven cases of ten were positive with royal blue signal in placent paraffin section in situ amplification,which indicated that placenta is probably one of the most important pathway of in

关 键 词：乙型肝炎病毒 慢性乙型肝炎 共价闭合环状DNA 母婴传播 原位聚合酶链反应 

分 类 号：R512.62[医药卫生—内科学] R714.2[医药卫生—临床医学]