乙型肝炎人源化小鼠模型的研制及动物实验的研究

Because of the lack of proper animal models, it is difficult for us to study the infection of HBV and the related immune responses and liver inflamation in vivo. Recently, some investigations indicated that the humanized mice were prepared by transplanting the human hepatic progenitor cells (HPCs) and CD34+ hematopoietic stem cells (HSCs) into the immunodeficient mice strain BALB/c-Rag-/- Il2rg-/- with AFC8 expression, which could be infected with HCV and produced the HCV-specific T cell responses. However, in the humanized mice the engraftment and differentiation levels of human liver cells were low, which limit the replication of virus. But several studies have showed that the immunodeficient NOD-scid Il2rg-/- (NSG) mice strain could support highly the engraftment of CD34+ HSCs. The aims of present study are (1) to prepare AFC8 transgenic NSG mice, and to optimize the conditions of apoptosis induction to enhance the engraftment levels of CD34+ HSCs and HPCs in the transgenic mouse. (2) to transplant the CD34+ HSCs incubated by cytokines and the HPCs transfected with miR-122 mimius into the newborn AFC8/NSG mice for improving the differentiation levels of CD34+ HSCs and liver cells in the transgenic mice. (3) to study the immune responses and hepatic inflammation in the humanized mice with HBV infection using molecular biological methods and immunological assays to provide the animal model for investigating the immune responses and hepatitis in human bodies with HBV.

因缺乏合适的动物模型，HBV感染及其引起的免疫反应和肝脏炎症研究受到限制。最近研究显示，将人肝祖细胞和造血干细胞共移植入AFC8转基因的BALB/c-Rag-/-Il2rg-/-而制备的人源化小鼠支持HCV感染，并产生HCV特异性的T细胞反应。但是，人的细胞在该小鼠体内的植入和分化水平较低。已有研究表明，NSG小鼠对人细胞植入具有较高的支持率。为此，本项目拟通过（1）制备表达AFC8的转基因小鼠AFC8/NSG、优化该转基因小鼠肝细胞凋亡条件，提高人的造血干细胞和肝细胞在该小鼠体内的植入水平；（2）将细胞因子孵育的造血干细胞和携带miR-122模拟物的肝祖细胞移植入转基因小鼠，提高人的造血干细胞和肝细胞在转基因小鼠体内的分化水平；（3）对人源化小鼠接种HBV，借助分子生物学、免疫学方法研究HBV感染引起的人源化小鼠免疫反应及肝脏炎症，为人感染HBV所致的免疫反应和肝脏炎症提供动物实验条件。

项目原计划通过研制一种人源化小鼠模型来研究乙肝病毒感染及其引起的免疫反应和肝脏炎症问题，但因执行难度太大而受阻。因此，项目现尝试借助其它模型继续研究上述问题。众所周知，乙肝病毒感染具有噬肝性特点。而研究显示，小鼠白蛋白基因启动子/增强子与腺病毒颗粒都能调节基因在肝细胞内特异性表达，利用1.3拷贝HBV基因组制备的转基因小鼠支持完整的病毒颗粒复制。因而， 我们将1.3拷贝HBV基因组分别插入到真核表达载体的小鼠白蛋白基因启动子/增强子下游和腺病毒载体上构建重组质粒，为制备肝脏特异性表达HBV的转基因小鼠提供材料。体外转染表达的结果表明小鼠白蛋白基因启动子/增强子提高了HBV胞膜蛋白的表达水平，且显著提高了分泌到细胞外的HBsAg水平。这些结果为该载体的体内表达提供了重要的参考数据。目前临床上的慢乙肝患者一般是由乙肝病毒携带者转变而来，患者肝脏受到免疫损伤。HBV转基因小鼠是日前研究HBV耐受的常用动物模型。已有实验结果显示，一定的免疫刺激能够打破HBV转基因小鼠的HBV免疫耐受状态，触发HBV特异的免疫反应。因此，为促进慢性乙型肝炎的发病机制与治疗策略研究，我们利用CD40的激动型抗体（佐剂）、TLR7 激动剂（佐剂）和HBsAg adw2的天然突变体表位肽S208联合腹腔免疫HBV转基因小鼠，12天时在小鼠脾脏内检测到了少量的抗原特异性CD44+IFN-gamma+细胞，该结果为HBV免疫耐受的打破、以及由此引起的免疫反应带来了的肝脏炎症研究带来了希望。