母乳及其活性成份对乙型肝炎病毒感染性的抑制作用与机制研究

Infection of hepatitis B virus (HBV) is endemic in China, and mother-to-infant transmission of HBV is a main reason of the chronic HBV infections. Abundant data from clinical observations demonstrate that, regardless of lack of the immunoprophylaxis in the past or availability of the immunoprophylaxis measures nowadays, neonates and infants fed with human breast milk do not have additional risks for HBV infection, although HBV may be detected in the milk of mothers with positive hepatitis B surface antigen (HBsAg); the mechanism is unknown. Based on the evidence that human breast milk has broad spectrum of antiviral infection property and that infants fed with human breast milk have lower incidences of infectious diseases, we propose the hypothesis that human breast milk has the activity to inhibit the infectivity of HBV. Thus, HBV is not transmitted by breast milk feeding. Our previous investigations show that human breast milk can bind with HBsAg and the human lactoferrin is identified as the main active component for the bindings. With the primary hepatocyte culture system to support the replication of HBV in vitro, we performed the inhibitory test of viral infection and the preliminary data showed that human breast milk may significantly inhibits the infectivity of HBV. In the present study, we will further characterize the binding of human breast milk and lactoferrin with HBsAg, and study the inhibitory effect of human breast milk and lactoferrin on the infectivity of HBV and relevant mechanisms. The accomplishment of the present study will be of great importance to understand the mechanism of the lack of HBV transmission by breast milk feeding. This will be valuable to alleviate HBsAg-positive mothers’ anxiety for worrying about HBV transmission and to raise the rate of breast milk feeding in infants born to HBV-infected mothers.

乙型肝炎病毒(HBV)母婴传播是我国慢性HBV感染的重要原因。临床研究证明，无论在过去无免疫预防，还是现在采取预防措施，虽然乙型肝炎表面抗原(HBsAg)阳性母亲的乳汁存在病毒，但母乳喂养不增加新生儿HBV感染风险，其机制不明。根据母乳具有广谱抗病毒活性和母乳喂养新生儿感染病发生率低的特点，我们假设：母乳具有抑制HBV感染性的功能，故母乳喂养不传播HBV。我们前期研究发现，母乳能与HBsAg结合，并确定乳铁蛋白是其活性成份；HBV原代肝细胞培养和病毒感染抑制实验结果显示母乳能明显抑制HBV的感染性。本项目将综合利用病毒学（特别是HBV肝细胞原代培养）、分子生物学和蛋白质分析等技术，深入研究母乳以及乳铁蛋白与HBsAg结合以及抑制HBV感染性的特性及其机制。本研究对理解母乳喂养不传播HBV的机制，消除HBsAg阳性母亲母乳喂养的焦虑，提高HBV感染母亲子女的母乳喂养率有重要意义。

本研究根据乙型肝炎表面抗原（HBsAg）阳性即乙型肝炎病毒（HBV）感染产妇乳汁中含有病毒，但母乳喂养不增加婴幼儿感染风险这一背景，开展母乳是否具有抑制HBV感染性的研究。首先通过竞争抑制法，以牛乳和羊乳为对照，发现母乳全乳和母乳乳清能竞争性抑制血源和酵母重组HBsAg与抗-HBs结合，牛乳和羊乳几乎不能竞争结合，而母乳全乳和母乳乳清不能与抗-HBs结合，提示母乳能与HBsAg，且乳清经加热处理后仍具有其结合特性。通过Far-Western blotting方法，证实母乳乳清在分子量大约80kD的蛋白条带能与HBsAg结合。采用蛋白质谱分析技术，初步确定该蛋白条带为乳铁蛋白；进一步通过竞争抑制实验证明，重组和天然人乳铁蛋白能与HBsAg。利用稳定表达钠离子-牛磺胆酸共转运蛋白（NTCP）、适合于HBV培养的HepG2 细胞体系（HepG2-NTCP），以乙型肝炎免疫球蛋白（HBIG）为阳性对照，小牛血清为阴性对照，进行体外中和实验。结果显示母乳乳清和重组和天然乳铁蛋白，均能抑制HBV感染HepG2-NTCP细胞。..同时，我们进行了母乳进行HBV包膜溶解实验和HBV DNA释放实验，结果显示，母乳不具有溶解HBV包膜的特性，因此不能直接破坏HBV的完整性。.此外，我们定量分析了55对HBsAg阳性产妇的血清及其乳汁的病毒载量，即HBV DNA水平，其中11对来自HBeAg阳性母亲。结果显示，11对HBeAg阳性母亲标本，血清HBV DNA均阳性，平均滴度为5.69 ± 2.96 log10 IU/ml；6例（54.5%）乳清HBV DNA阳性，平均滴度为3.52 ± 0.48 log10 IU/ml；44对HBeAg阴性母亲标本，36例（81.8%）血清HBV DNA阳性，平均滴度为2.64 ± 1.09 log10 IU/ml；仅14例（31.8%）乳清HBV DNA阳性，平均滴度为2.48 ± 1.19 log10 IU/ml。因此乳汁中病毒水平明显低于血清水平。..综合本项目的所有研究结果，HBV感染母亲的母乳中存在病毒，但明显低于血清病毒水平；母乳能与HBsAg以及HBV结合，从而抑制病毒的感染性，其中的关键成份是乳铁蛋白。本研究阐明了HBV感染母亲乳汁中存在病毒，母乳喂养不增加HBV的母婴传播的机制，为HBV感染母亲的婴儿能母乳喂养提供了充分的科学根据。