先天感染华南鸭乙肝模型的建立并验证肝经引经药对3TC抗乙肝病毒疗效的影响

【作者】 杨书兰；

【导师】 郭兴伯；

【作者基本信息】 广州中医药大学 ， 中西医结合基础， 2001， 博士

【摘要】 乙型肝炎以易于慢性化为其特征，部分慢性肝炎持续多年，最终可发展为肝硬化及肝癌，目前尚无满意的治疗方法。鸭乙型肝炎病毒与人类乙型肝炎病毒同属嗜肝DNA病毒科，鸭乙肝模型近年来已成为筛选和评价抗-HBV药物的常规模型。但在国内尚未发现简单、实用、可行的建立先天感染鸭乙肝模型的方法，制约了抗乙肝药物的研究。 中西医结合治疗慢性乙肝，近几年已进行了多方面的探索与研究，但多停留于中西药简单的合用阶段。 本课题应用PCR及Dot-blot两种方法筛选建立先天感染鸭乙肝模型，从血清病毒学及病理学两方面对先天感染鸭乙肝模型与一日龄雏鸭感染所致的后天感染模型进行了比较，并利用先天感染鸭乙肝模型，研究了肝经引经药柴胡及《伤寒论》少阳病主方小柴胡汤对拉米呋啶抗乙肝病毒作用的影响，探索以中医理论指导中西医结合治疗慢性乙型肝炎的方法。 结果证明PCR法与Dot-blot法比较，有简单、快捷、敏感度高等优点，且可避免Dot-blot带来的放射性污染。但是PCR法所需费用高，易因交叉污染而导致假阳性结果，影响以后的实验。 已有的实验结果证明，先天感染鸭在两周内血清病毒滴度极高，两周后即下降至一个较低水平，并持续两年左右。我们的实验亦发现，相同剂量的药物，在雏鸭十七日龄时给药有效，而于二日龄时给药则无效。 先、后天感染鸭乙肝模型的比较结果显示，先天感染模型血清病毒滴度高，持续时间长。肝脏的病理改变亦有明显不同：先天感染鸭肝的炎性改变（包括围管浸润、枯否细胞增生、局灶性坏死）多集中于汇管区及中央静脉周围，肝细广州中医药大学98纽博士论文 作者：杨书兰 广州中医荧大学热带医学研究所胞的损害均较后天感染为重；而后天感染鸭肝多表现为广泛星点样坏死，大部分血管均出现围管浸润，但细胞病变较轻，在接种病毒后第28天左右，肝脏趋于正常。这一结果证明，先天感染鸭乙肝模型的病理变化更接近慢性、兔疫耐受性乙型肝炎病人的病理变化。 药物抗病毒实验的结果证明，拉米味吱在smglkgld剂量时，于先天感染二日龄雏鸭给药无效，于十七日龄雏鸭给药则有明显抑制血清病毒滴度的作用；柴胡不能降低血清病毒滴度，亦未能明显延缓拉米吠睫停药后的反跳；小柴胡汤能有效降低先天感染鸭的血清病毒滴度，亦有一定的延缓拉米味咬停药后反跳的作用。肝脏病理检查结果证明，给予拉米味陡后不会加重肝脏的炎性病变（与对照组相比p＞o．05卜细胞病变略有减轻：柴胡不能改善肝脏的病理改变（与对照组相比p＞o．05＊ 服用小柴胡汤后，鸭肝细胞出现严重的溶解现象，炎症改变亦明显加重（与对照组相比p＜o．01卜 由此可见，拉米吱陡的抗病毒作用与机体的兔疫清除作用关系不明显，而中药小柴胡汤则是通过免疫调节作用来清除病毒的。柴胡合用拉米吠陡后，肝脏的炎性改变未见好转（与对照组相比p＞o．05卜 但细胞损伤明显减轻，基本趋于正常；小柴胡汤合用拉米吠陡治疗过程中，肝脏病理改变亦明显好转。而且柴胡及小柴胡汤与拉米吠唆合用后，鸭肝细胞中的网状纤维亦较对照组及单用拉米吠陡组轻微减少。说明肝经引经药柴胡或少阳病主方小柴胡汤与拉米吠陡合用，有可能通过协同作用杀灭肝细胞内病毒而起到减少肝细胞损伤、控制肝纤维化的作用。 多脏器病毒检测实验结果证明，乙肝病毒不仅存在于鸭肝脏中，而且存在于脾、肺、肾、胰、生殖器、脑等多个脏器中。这可能是乙肝病毒难于清除的原因之一。因此乙肝病 2广州中医药大学98级博士论文 作者：杨书兰 广州中医茹大学热带医学研究所人的治疗不能局限于某一脏器，而应从整体上、从脏器的相互关系上、从免疫调节等各方面进行论治。更多还原

【Abstract】 Hepatitis B virus(HBV) can cause chronic hepatitis, hepatocirrhosis and hepatocarcinoma. There is no specific curative medicine for it so far.Both duck hepatitis B virus(DHBV) and HBV belong to the family Ilepadnaviridae. DHBV model has already been used as the routine model in experiment for screening and evaluating anti-hepatitis medicine in recent years. However because there has been no sim.ple, applicable and practical method to establish innated-infected DHBV model, which is the main obstacle for the research of anti-hepatitis medicine. In recent years, treatment of chronic hepatitis B combining herbs and western medicine has been studied in many aspects. But most of the studies were limited in simple superimposing of herbs and western medicine. The study includes the following three approaches: firstly,pre-natally infected hepatitis B virus ducks were selected using the methods of PCR and Dot-blot, of which the sensitivity were compared. Secondly, the differences between pre-natally-infected and postnatally -infected DHBV models were testified. Thirdly, on pre-natally- infected DHBV model, the synergistic effects of Bupleurum root (the meridian-directing herb of liver based on TCM theory) and Minor Decoction of Bupleurum (main prescription of ShaoYang channel in Shang Han Lun) and Lamivudine on DHBV were studied in an attempt, by which to search for the method of treating chronic hepatitis B by combining herbs and western medicine based on theory of TCM. We found PCR method was more sensitive, more simple and more rapid than Dot-blot. It can avoid the radiative pollution caused by Dot-blot, yet PCR was more expensive and a fake positive result would be found due to cross- pollution. We also found if we used PCR to select the ducks ,and gave medicine to them on the second day, the dose should be increased. Because the peak of viremia in pre-natally-infected model lasted for two weeks and then decreased to a lower level and last for about two years111. Comparison with the postnatally-infected model, the level of DHBV of congenitally-infected model was higher and more stable. he pathological change of liver was more similar to that of the human-being with chronic hepatitis B. Lamivudine and Minor Decoction of Bupleurum had a significant inhibitive effect on DHBV-DNA in pre-natally- infected model. The results of pathology of liver showed that Lamivudine could clear DHBV directly while Minor Dectoion of Buplcurum cleared virus through immunoreaction mechanism. Bupleurum Root had no significant inhibitive effect on DHI3V-DNA. The herbs can regulate human’s immune system to synergize anti-DHBV effect of Lamivudine in liver so as to delay the relapse and alleviate the pathology of liver. Furthermore, combination of herbs and Lamivudine can inhibit fibrosis of liver. The results of the experiment have demonstrated that DHBV can invade several organs such as liver, spleen, brain, lung, pancreas, genitalia, kidney, which is one of the reasons why it抯 difficult in clearance of HBV or DHBV. Therefore the treatment of HBV shouldn’t only focus on liver. Various factors should be taken into consideration in treatment strategy, such as the mutual relations between organs and immune mechanism, etc.更多还原