乙型肝炎病毒转基因小鼠免疫耐受状态及血液生物化学指标的研究

Study on the immune tolerance status and blood biochemical index of hepatitis B virus transgenic mice

目的了解HBV转基因鼠HBV基因的复制表达和免疫耐受状态，为探讨乙型肝炎发病机制和抗HBV新药评价提供可靠的参考依据。方法选取遗传背景相同的SPE级HBsAg阴性非转基因鼠和转基因鼠。化学发光法检测HBsAg、HBeAg、HBVDNA，ELISA检测前S1、HBcAg，肝组织行病理学检查，免疫组织化学染色检测不同时期转基因鼠肝HBsAg表达，流式细胞仪检测小鼠淋巴细胞增殖情况，酶联免疫斑点检测（ELISPOT）分泌IFNy的T淋巴细胞斑点数，双色免疫荧光法检测脾细胞悬液和脾树突状细胞（DC）中Toll样受体（TLR）2和TLR9的表达。数据行t检验和F检验。结果HBV转基因鼠可复制表达HBsAg、前S1、HBeAg、HBcAg和HBVDNA，而抗-HBs、抗-HBc、抗-HBe均阴性；肝组织无明显病理改变，肝细胞中HBsAg在胞质表达，HBcAg在胞核表达。HBsAg刺激后，HBV转基因鼠T淋巴细胞增殖能力为（697．6±67．3）cpm，显著低于非转基因鼠的（1315．5±191．6）cpm。经HBsAg刺激后，HBV转基因鼠脾细胞分泌IFNγ的T淋巴细胞斑点数为8．25±1．10，低于非转基因鼠的28．50±4．21（F=155．967，P=0．000）。HBV转基因DC表达CD11c^＋、TLR2和TLR9与非转基因鼠比较，差异无统计学意义（均P〉0．05）。在18日龄胎鼠和1日龄仔鼠肝组织观察到HBsAg表达。结论HBV转基因鼠有HBV相关抗原表达，并对HBV相关抗原存在免疫耐受，其先天和获得性免疫功能均正常，类似于人类慢性HBV无症状携带者。HBV转基因鼠是比较理想的动物模型。

Objective To research the hepatitis B virus （HBV） replication and immune tolerance status of transgenic mice for elucidating the pathogenesis of hepatitis B and evaluating new drugs against HBV. Methods SPE grade HBsAg negative nontransgenic and transgenic mice with the same genetic background were recruited in this study. HBsAg, HBeAg and HBV DNA were detected by chemiluminescent method. Pre S1 and HBcAg were detected by enzyme linked immunosorbont assay （ELISA）. Liver pathology was examined and HBsAg expressions at different stages were determined by immunohistochemical staining. The lymphocyte proliferation of mice was detected by flow cytometry and interferon （IFN） y-producing T lymphocytes was determined by enzyme linked immunospot （ELISPOT）. The expressions of Toll-like receptor （TLR）2 and TLR9 in splenocyte suspension and splenic dendrite cells （DC） were determined by double-labeling immunofluorescence. The data were analyzed by t test and F test. Results HBsAg, preS1, HBeAg, HBcAg were expressed and HBV DNA was replicated in HBV transgenic mice, while anti-HBs, anti-HBc, and anti-HBe were all negative. There were no obvious pathological changes in liver tissues. HBsAg was expressed in cytoplasm and HBcAg in nucleus of hepatocytes. After stimulated with HBsAg, T lymphocyte proliferation capacity of HBV transgenic mice was （697.6±67.3） cpm, which was much lower than that of nontransgenic mice [（1315. 5±191. 6） cpm]. The number of spot forming cells of IFNγ-producing splenocytes from transgenic mice after HBsAg stimulation was 8. 25 ±1. 10, which was obviously lower than that of nontransgenic mice （28.50±4.21） （F=155. 967, P=0. 000）. The expressions of CD11c^＋ , TLR2 and TLR9 on DC from both HBV transgenic and nontransgenic mice were not different significantly （all P〉0.05）. The HBsAg expressions in liver tissues were observed in 18-day-old fetal mice and 1-day-old newborn mice. Conclusions The HBV transgenic mice can express HBV-related antigens, and are immune tolerant to the antigens. The innate and acquired immunity of the HBV transgenic mice are normal, which is similar to chronic asymptomatic HBV carriers of human. Therefore, HBV transgenic mouse is an ideal animal model.