【作者】 朱明添；

【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， 2012， 博士

【摘要】 经过多年实验研究、临床研究、反复筛选、不断优化,本研究采用叶下珠、黄芪、三七、甘草等数种中药组成了叶下珠复方,并拟将其开发为新一代的抗乙肝病毒的新药。在进行叶下珠复方的临床研究前,我们完成体外抗乙肝病毒(HBV),对HBV转基因小鼠免疫病理模型的干预作用的实验研究,验证了其实验治疗的有效性。在体外抗HBV的研究中,我们选择了2.2.15细胞作为体外模型,HBsAg、HBeAg作为药物效果的筛选靶标,通过MTT法测定药物的细胞毒性浓度,并相应计算出治疗指数来评价药物疗效。结果显示,叶下珠复方在2.2.15细胞中对HBsAg分泌抑制的治疗指数为8.4.因此,叶下珠复方在体外能高效、低毒的抑制HBV的分泌HbsAg和HBeAg。叶下珠复方中、大剂量及ADV在Tg鼠血清HBsAg阴转率分别在给药后第10天、第21天、停药后3天进行比较,差异均无显著性意义,ADV具有减少HBV Tg鼠血清HBV-DNA载量的作用。但停药3天有反跳。叶下珠复方大、中剂量组均有降低HBV Tg鼠血清HBV-DNA载量的作用,尤其以大剂量组效果明显,虽然没有减少到ADV组水平,但治疗前后差异有显著性意义,且在停药后无明显反跳现象,说明叶下珠复方作用持久、平稳。模型组Tg鼠肝脏病理改变类似于临床轻度肝炎表现,随着叶下珠复方剂量的增大,炎症得以缓解。大剂量组的肝板排列整齐,无纤维组织增生,但中央静脉周围轻度围管浸润。叶下珠复方中、大剂量和ADV均能减少肝组织中HbcAg表达,尤其大剂量组明显。叶下珠复方能够上调CD3+、CD4+、CD4+/CD8+,下调CD8+T细胞比值水平,具有正向T淋巴细胞免疫调节作用,尤其以大剂量组效果明显。ADV也具有提高HBV转基因小鼠细胞免疫功能(如CD3+百分比),但作用相对叶下珠复方弱。运用大、中、小剂量叶下珠复方和ADV均能提高HBV转基因小鼠血清IL-2、TNF-γ含量,与模型组相比有显著的差异。且运用大、中剂量叶下珠复方治疗后IL-2、TNF-Y水平已高于正常对照组。叶下珠中剂量组CD3+、TNF-γ与给药前后HBV-DNA载量的减少呈显著正相关,叶下珠复方大剂量组CD4+、CD8+、CD4+/CD8+的变化与给药前后HBV-DNA减少呈显著正相关。叶下珠复方联合阿德福韦酯(ADV)治疗拉米夫定耐药肝炎(LRHB)52周,ALT复常率、HBV-DNA阴转率、HBeAg阴转率均显著优于单纯使用ADV组,且差异显著(P<0.05或P<0.01)。治疗52周,叶下珠复方联用ADV完全应答率(CR)为25%,显著高于ADV组(10%),X2=9.07,P<0.01。叶下珠复方联合ADV组治疗52周,患者CD4+百分比和NK细胞均增加,CD3+百分比增加,而两组CD8+百分比减少,并与HBV-DNA载量的减少呈显著正相关。HBV特异型CD8+T细胞用于破坏感染肝细胞,能清除肝内HBV、从而减少细胞内HBV数目,说明叶下珠复方联合ADV能更好改善LRHB患者T淋巴细胞亚群的异常及提高NK细胞的活性,提高抗病毒的疗效。同时能调节免疫功能,使免疫低下或免疫紊乱的调整到正常,显示出较好的抗HBV效果。更多还原

【Abstract】 For many years of experiment and clinical research, repeatedly selection and continuous optimization, our study use Phyliathu urinaria Linn (PUL), Astragalus membranaceus, pseudo-ginseng, liquorice, etc.to compose the capsule of Phyliathus Complex (PC).We hope to explore a new generation of traditional Chinese herbal combination to treat chronic hepatitis B.Before the clinic study, we have accomplished a series of experiment research of anti-HBV in vitro,and an experiment research about the intervention of HBV transgenic mouse immunopathogenesis model.We proved the efficiency of treatment.In the research of anti-HBV in vitro,we choose2.2.15cell as a vitro model,HBsAg、 HbeAg as drug effect of the preliminary screening targets.Though MTT method measure cell toxicity concentration of medicine,and calculate therapeutic index to evaluate medicine efficiency. Results show that, the therapeutic index of Phyliathus Complex inhibiting HBsAg in2.2.15cell is8.4.So Phyliathus Complex has high-efficiency and low-toxin to inhibit HBsAg and HBeAg in vitro.The rate of HBsAg negative-inverted with using Phyliathus Complex of medium-dose, high-dose and ADV in Tg mouse are compared respectively at10d,21d, after without drug3d. There is no significant difference.ADV has an effect of reducing serum HBV-DNA load in HBV Tg mouse.But after without drug3d, it has a bounce.Phyliathus Complex of medium-dose, high-dose has an effect of reducing serum HBV-DNA load in HBV Tg mouse. Specially, high-dose has a significant effect. HBV-DNA load can’t reduce to the level of ADV group, but there is a significant difference before and after treatment, and don’t appear bounce after without drug. We can see that Phyliathus Complex have a stable and durable effect. In model group,Tg mouse liver pathological changes is similar to the expression of clinical mild hepatitis, with increasing the dose, inflammation can be relieved. The hepatic plates of high-dose group arrange appropriately, there is no fibrous tissue hyperplasia, but perivascular inflammatory infiltrates mildly. Phyliathus Complex of medium-dose, high-dose and ADV all can decrease HBcAg expression in liver issue, especially in high-dose group.Phyliathus Complex can up regulate CD3+、CD4+、CD4+/CD8+, and down regulate level of CD8+T cell, has a positive immune regulation for T cell lymphocyte, specially in high-dose group.ADV also can improve immune function of HBV transgenosis mouse cell (such as percentage of CD3+), but the effect of ADV is lower than Phyliathus Complex.Using Phyliathus Complex of high, medium,small dose and ADV can all increase serum IL-2and TNF-r content in HBV transgenosis mouse,there is a significant difference compared with control group.And after using Phyliathus Complex of high-dose and medium-dose, IL-2and TNF-γ content is higher than normal group.In Phyliathus Complex of medium-dose group, CD3+、TNF-γ have a positive correlation with reduction of HBV-DNA load before and after treatment.In high-dose group, changes of CD4+、CD8+、CD4+/CD8+have a positive correlation with reduction of HBV-DNA load before and after treatment.Phyliathus Complex combining with ADV treat Lamivudine resistant hepatitis B (LRHB) for52weeks.ALT recovery rate, the rate of HBV-DNA and HBeAg negative-inverted are all higher than single AVD group, and there is a significant difference (P<0.05or P<0.01).After52weeks treatment, the complete response rate of Phyliathus Complex combining with ADV is25%,remarkable higher than ADV group(10%), X2=9.07,P<0.01.Using Phyliathus Complex combining with ADV group after52weeks treatment, CD4+, CD3+percentage and NK cell of patients all increase, but CD8+percentage of two group all decrease, and has a positive correlation with reduction of HBV-DNA load. HBV specific type CDg+T cells destroy infective liver cell, eliminate HBV in liver, thus reduce amount of HBV in cells.It shows, Phyliathus Complex combining with ADV can improve abnormal situation of T cell lymphocyte in LRHB patients, and increase NK cell activity, enhance antiviral efficiency. Meanwhile, Phyliathus Complex combining with ADV also can regulate immune function; make low or abnormal immune to normal situation,so it shows a better anti-HBV effect.更多还原