HBx蛋白和表皮生长因子受体在HBV宫内感染中作用机制的研究

Many studies have shown that the main route of HBV intrauterine infection is transmittion through the placenta. However, the molecular mechanism remains unclear. In our studies over the past 10 years we have confirmed the placental pathway of HBV intrauterine transmission, and have developed measures to effectively block this transmission.On Foundation of National Natural Science(The study of action of cell apoptosis and HBxAg in HBV intrauterine infection, 30872220),In a recent study we transfected trophoblasts with a HBx expression vector and observed HBxAg expression in trophocyte in the state of the high infectivity , the HBx protein was located in the cytoplasm and nucleus. The HBx protein was observed to inhibit apoptosis in the cultured transfected cells by activating PI3K/pAkt,this makes trophoblast of HBV infection longevity,and provide lurking places for the virus, make virus immune escape. Therefore the HBx protein play a vital role in the molecular mechanism of HBV intrauterine infection. We propose to study epidermal growth factor receptor (EGFR) expression after HBx gene transfection of trophocytes; to detect the role of HBx protein in EGFR promoter activation using an chloramphenicol acetyltransferase (CAT) assay. Secondly,the HBVDNA of HBx-deficient and wild-type HBV will be used to transfected trophocytes, we will detect HBxAg、HBsAg and HBVDNA in both supernatant and cells using technologies such as real-time quantitative PCR, Southern blot and sucrose density gradient analysis.We propose that our studies will show: 1. HBx produces the anti-apoptotic state in HBV infection placenta through the EGFR/PI3K/Akt due to increased levels of EGFR pathway; 2. the regulatory regions of EGFR gene are the targets of HBx protein trans activities; 3. HBx protein can promote hepatitis B virus replication in placental cells. The results of these experiments can provide a solid basis for to investigate new blocking agents from new perspective of inhibiting the expression of HBx protein.

大量研究表明HBV宫内感染的主要途径是经胎盘传播，但分子机制仍不清楚。我们在10余年HBV宫内感染研究的基础上，发现HBV高复制状态下HBx蛋白通过激活PI3K/pAkt抑制滋养细胞凋亡使病毒发生免疫逃逸，证实HBxAg在HBV宫内感染中起重要的作用。本课题通过HBx基因转染滋养细胞进行表皮生长因子受体（EGFR）表达的研究；通过氯霉素乙酰转移酶（CAT）检测EGFR启动子对HBx的作用；利用HBx缺失型和野生型HBVDNA转染滋养细胞，实时定量-PCR、Southern blot及蔗糖密度梯度分析等检测上清液和细胞HBVDNA。明确HBxAg在胎盘细胞能激活EGFR并且EGFR是HBx反式作用靶位；明确HBxAg是胎盘滋养细胞中HBV复制的重要因素。首次证实HBx蛋白通过多种机制促进HBV感染胎盘和宫内感染的发生。以期提出从抑制HBx蛋白表达角度的HBV 宫内阻断新措施提供坚实的理论依据

宫内感染机制和阻断措施的研究在控制我国乙肝流行非常重要。本项目主要研究HBx蛋白和表皮生长因子受体在HBV宫内感染中作用机制的研究。发表论文共4篇。其中2篇中文核心期刊，2篇被SCI收录。获2016年陕西省科技进步二等奖（S2016JLJS0469）。培养硕士研究生3名。. 1.通过HBx 真核表达载体pcDNA3.1-x 体外转染JEG-3 及HTR-8 细胞，发现转染组细胞凋亡明显低于对照组（P<0.05）；转染组细胞内PI3K，p-AKT 的表达明显高于对照组 (P<0.05)。并且通过siRNA 敲低HBx 实验验证以上结果。证实HBxAg/PI3K/p-AKT 途径是HBV 抑制细胞凋亡的可能机制。.2.研究发现HBx过表达能增加EGFR和AKT的磷酸化水平从而抑制滋养细胞凋亡。敲低HBx后验证了以上结果。构建EGFR稳定表达的滋养细胞发现PI3K和p-AKT表达升高，抑制滋养细胞细胞凋亡。证实HBx对PI3K/p-AKT/抑制细胞凋亡作用可能是通过激活p-EGFR实现的。.3.我们通过分别加入PI3K 抑制剂LY294002、AKT 抑制剂MK2206，发现HBX 组的早期凋亡率明显低于HTR-8-pcDNA3.1-HBX+PI3K抑制剂组、HTR-8-pcDNA3.1-HBX+AKT 抑制剂组和HTR-8-pcDNA3.1细胞组（p<0,05）。验证了细胞PI3K/ p-AKT 途径与滋养细胞凋亡有直接关系。进一步证实HBx激活PI3K/p-AKT/而抑制细胞凋亡。.4. 通过1.0HBV及HBX缺失突变型1.0HBV质粒构建，证实上清液中HBX蛋白对HBV DNA复制影响。收集细胞， 1.0HBV组HBeAg的表达和HBVDNA复制水平均显著高于HBX缺失突变组 (P<0.05)，证实HBx在滋养细胞中对HBV复制的重要作用。