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目的检测HBV对胎盘、胎肝和滋养层细胞的感染情况,探讨HBV的宫内感染机制。方法研究对象包括20例孕妇胎盘组织、6例引产胎儿胎肝组织及体外培养的胎盘滋养层细胞。ELISA法检测孕妇外周血、胎儿脐血和6个月婴儿外周血HBV标志物;荧光定量PCR法检测血清和滋养层细胞中的HBV DNA;免疫组织化学法和免疫荧光法检测胎盘、胎肝组织及滋养层细胞中HBV标志物的表达;末端脱氧核糖核酸转移酶介导的缺口标记法(TUNEL)检测胎盘和滋养层细胞凋亡情况。结果孕妇血清HBV DNA水平与胎儿脐血HBV DNA水平相关,脐血HBV DNA阳性者其母血HBV DNA>1.0×10~7拷贝/mL;6例胎盘组织和3例引产胎儿胎肝组织中可见HBsAg免疫组织化学染色阳性细胞,其中1例胎肝组织中发现HBcAg阳性细胞;体外培养滋养层细胞与HBV DNA阳性血清共孵育后,可检测到HBsAg的表达,亦可检测到HBV DNA。体内和体外实验均检测到HBV感染后滋养层细胞凋亡呈增加趋势,且胎盘细胞的凋亡与脐血HBV DNA水平相关。体外实验结果显示,随感染时间的延长,滋养层细胞凋亡呈增加趋势。6个月后,12例新生儿有1例血清HBsAg、HBeAg和抗-HBc阳性,6例抗-HBs阳性。结论HBV宫内感染的机制可能是通过HBV感染胎盘屏障而使胎儿发生HBV宫内感染。HBV在胎儿组织器官内的定位和复制可能是新生儿发生慢性HBV感染的重要因素。滋养层细胞凋亡可能是胎盘屏障阻断HBV宫内传播的一种保护性机制。
Objective To detect the infection of HBV in placenta, fetal liver and trophoblast cells and to explore the mechanism of HBV intrauterine infection. Methods The study included 20 cases of pregnant women placenta tissue, 6 cases of induced fetal fetal liver tissue and cultured in vitro trophoblast cells. Serum and trophoblast HBV DNA were detected by real-time PCR and peripheral blood mononuclear cells (PBMCs) in pregnant women, fetal umbilical cord blood and 6-month-old infants. The placenta and fetal liver tissues were detected by immunohistochemistry and immunofluorescence And trophoblast cells HBV markers; TdT-mediated nick end labeling (TUNEL) detection of placental and trophoblast apoptosis. Results The level of serum HBV DNA in pregnant women was related to the level of HBV DNA in fetal umbilical cord blood. HBV DNA level> 1.0 × 10 ~ 7 copies / mL in umbilical cord blood was positive for HBV DNA; 6 cases of placenta tissue and 3 cases of induced fetus HBsAg immunohistochemical staining of positive cells, of which 1 case of fetal liver tissue found in HBcAg-positive cells; cultured in vitro trophoblast cells incubated with HBV DNA-positive serum can detect the expression of HBsAg, HBV DNA can also be detected. Both in vivo and in vitro experiments showed that the apoptosis of trophoblast cells showed an increasing trend after HBV infection, and the apoptosis of placental cells correlated with the level of HBV DNA in cord blood. In vitro experiments showed that with the extension of infection time, trophoblast apoptosis showed an increasing trend. Six months later, one of 12 neonates had positive serum HBsAg, HBeAg and anti-HBc, and 6 anti-HBs positive. Conclusion The mechanism of intrauterine infection of HBV may be that intrauterine infection of HBV occurs in fetus through HBV infection of placental barrier. Localization and replication of HBV in fetal tissues and organs may be an important factor in neonatal chronic HBV infection. Trophoblast apoptosis may be a protective mechanism by which the placental barrier blocks the intrauterine transmission of HBV.