【作者】 向志超；

【导师】 王新华；

【作者基本信息】 广州中医药大学 ， 中医临床基础， 2009， 博士

【摘要】 肝纤维化,是一种发生于慢性肝病后的可逆性损伤愈合反应,各种慢性肝病向肝硬化发展的所必经病理过程。目前,对于许多慢性肝病的治疗尚无理想的针对治疗措施,如乙型肝炎病毒所导致的病毒性肝炎。从病理变化来说,肝纤维化是可逆的,而肝硬化则是不可逆的,再加上我国是病毒性肝炎流行的高发区,据最近统计资料显示,我国目前有肝病患者已达2000多万,肝纤维化在我国的发病率较高,是我国人民必须严肃对待的一个健康问题。因此,寻找防治肝纤维化的有效方法和有效药物,是我国人民的迫切要求,具有深远的意义和重大的社会价值。乙型肝炎是由乙型肝炎病毒（HBV）感染所致,全世界约有3亿人是HBV携带者,而HBV携带者发生肝纤维化、肝硬化和肝癌的危险性比非携带者高出上百倍。我国是乙肝高发区,约有1.2亿HBV携带者。但是由于乙型肝炎的临床表现并不明显,再加上大多数病人并不重视而导致迁延失治,往往会让病情发展为慢性肝炎和肝纤维化。慢性肝炎（HBV所致）的存在是引发肝纤维化的重要原因,单单治疗肝纤维化是无法使之完全治愈的,就算治疗得当也无法排除复发的危险。因此,抗慢性肝炎肝纤维化应作为一个整体进行治疗,方是彻底治愈的有力保证。国内外在对慢性肝炎及肝纤维化的机理研究方面已取得了一定进展。但是在其临床治疗领域,却依然未能取得明显的进步。加上目前临床现有的药物,其疗效并不够理想,而且有些药物普遍存在副作用较大,费用较高的缺点。因此国内外的研究者都在努力寻找疗效确切、价格低廉、毒副作用小的防治肝纤维化药物,以便能阻止慢性肝炎进一步发展,演变为肝纤维化的进程,最终治愈大多数慢性肝炎及肝纤维化。因此本课题以加味三仁汤作为实验药物,展开针对慢性肝炎及肝纤维化的研究,拟采用免疫组化、细胞培养技术、流式细胞术、分子生物学方法（RealTime-PCR技术）等技术对鸭乙肝、大鼠免疫性肝纤维化、HSC细胞以及2.2.15细胞的影响等方面探讨其抗慢性肝炎及抗肝纤维化的作用及机理,为该方进一步的临床及开发应用提供实验依据。1.利用DHBV阳性绿头鸭作为研究对象,观察加味三仁汤对其血清中DHBV-DNA水平的影响。结果显示:模型组鸭血清中DHBV-DNA水平升高,与正常组比较有显著性差异（P＜0.01）,加味三仁汤水提液大剂量、小剂量以及大剂量醇提液对DHBV-DNA均有明显的抑制作用（P＜0.05或P＜0.01）,而加味三仁汤的小剂量醇提液则没有显著的抑制DHBV-DNA作用。2.以加味三仁汤作用于2.2.15细胞,观察其对2.2.15细胞HbsAg及HbeAg分泌的影响。结果显示,浓度为1、0.5、0.25、0.125、0.625、0.3125mg/mL的加味三仁汤水提液对2.2.15细胞HbsAg和HbeAg分泌抑制率分别为85.36%、79.72%、75.81%、69.65%、62.14%、30.56%以及90.17%、83.26%、76.34%、46.86%、20.32%、0.36%。药物剂量与HbsAg和HbeAg分泌抑制率呈现递增关系,其中随着剂量增加,其中药物对HbsAg的抑制更为明显。3.观察加味三仁汤对2.2.15细胞HBV-DNA表达的影响。结果表明,加味三仁汤具有明显的抑制2.2.15细胞HBV表达的作用,其中1 mg/mL、0.5 mg/mL及0.25 mg/mL浓度的加味三仁汤水提液效果更优于阳性药物。4.通过对DMN肝纤维化大鼠模型,观察加味加味三仁汤对肝组织肝HA、LN、IVC、PCIINP及HyP的影响。模型组及各给药组血清HA、LN、IVC、PCⅢ及HyP的水平均升高,与正常组比较,有显著性差异（P＜0.01）。加味三仁汤水提大、小剂量组与模型组比较,血清HA、PCⅢ、LN、CIV及HyP水平均明显下降（P＜0.05或0.01）,且随剂量增加效果也相应增强;加味三仁汤醇提大剂量组与模型组比较,血清LN、IVC、PCⅢ及HyP的水平有一定程度下降,但无显著意义（P＞0.05）。加味三仁汤醇提小剂量组与模型组比较,血清PCⅢ、CIV、HyP水平有下降,但无显著意义（P＞0.05）。鳖甲软肝片组血清HA、PCⅢ、CIV水平较模型组无显著差异（P＞0.05）。5.通过对DMN肝纤维化大鼠模型,观察加味三仁汤对DMN肝纤维化大鼠肝组织形态学的影响:（1）HE染色;（2）α-SMA免疫组化;（3）Ⅰ型胶原免疫组化。本实验表明,从肝小叶破坏、假小叶形成、胶原纤维增生等肝脏结构的客观改变出发,认为加味三仁汤可减轻肝纤维化的程度,改善肝脏结构,促进ECM降解,从而治疗实验性肝纤维化。6.观察加味三仁汤对大鼠肝组织TGFβ₁及PDGF-B表达抑制的影响。结果显示模型组及各给药组血清TGF-β₁及PDGF表达增加,与正常组比较,有显著性差异（P＜0.01）。加味三仁汤水提液组均能显著抑制TGF-β₁和PDGF的表达（P＜0.05）,且优于阳性药物,其中尤以加味三仁汤水提大剂量组最为明显（P＜0.01）,且随剂量增加效果也相应增强;加味三仁汤醇提小剂量组剂量组与模型组比较,对于TGF-β₁和PDGF表达抑制的影响有显著意义（P＜0.05）。加味三仁汤醇提组与模型组比较,对血清TGF-β₁的影响无显著意义（P＜0.05）。7.观察加味三仁汤对肝纤维化大鼠血清TNF、IL-1及IL-6的影响。结果提示,加味三仁汤水提液组均具有显著降低大鼠血清中TNF-α、IL-1以及含量的作用（P＜0.05或P＜0.01）。其中水提液大剂量组的效果更是优于阳性药物组。而加味三仁汤醇提大剂量组对大鼠血清中的TNF-α和IL-1有明显的降低作用,但对IL-6则无明显作用。而小剂量醇提液对大鼠血清中的TNF-α、IL-1和IL-6则无明显作用。8.探讨加味三仁汤对HSC-T6 TIMP-1和MMP-2表达的影响。结果显示40mg/mL和20mg/mL浓度的加味三仁汤能有效降低TIMP-1和MMP-2mRNA的水平,并随着浓度降低而减弱。9.观察大鼠肝组织和HSC中的Fas/FasL表达与加味三仁汤的干预作用。实验发现,加味三仁汤能明显上调大鼠肝组织及HSC Fas/FasL基因mRNA的表达,而且其水提液对Fas/FasL的上调作用均优于阳性药物组。10.探讨体外培养HSC凋亡与加味三仁汤的干预作用。实验提示:加味三仁汤和丹参注射液均可诱导HSC凋亡,其中加味三仁汤水提液40mg/ml和20mg/ml剂量组细胞凋亡明显,并优于丹参注射液组。此外,加味三仁汤组对HSC的凋亡促进还呈剂量依赖。11.探讨加味三仁汤对肝纤维化大鼠组织纤维酶原激活剂抑制剂、组织型纤维酶原激活剂的影响。结果显示:与细胞对照组比较,加味三仁汤可调节uPA及PAI-1的表达。其中浓度为40mg/mL以及20mg/mL的加味三仁汤水提液对HSC-T6细胞uPA及PAI-1的的表达调节优于其它各组。而在大鼠血清uPA及PAI-1的表达抑制中,加味三仁汤水提液组均优于其他各组。更多还原

【Abstract】 Liver fibrosis,is a reversible reaction after the chronic liver disease,and the inevitable development of the pathological process for the chronic liver disease to cirrhosis.At present, there hasn’t ideal treatment for some chronic liver diseases.such as the virus hepatitis which caused by hepatitis B virus hepatitis. From the pathological change,the liver fibrosis is reversible, and liver cirrhosis is irreversible.China is a high incidence of viral hepatitis epidemic.According to recent statistics,it shows that China has more than 20 million patients with liver disease.The IN of liver fibrosis is very high in our country,so it is a serious healthy problem that the national people must treat to.Therefore,searching for an effective prevention and treatment, for liver fibrosis,is an urgent requirement of national people and have far-reaching significance and important social value.HB is caused by the hepatitis B virus(HBV).There have about 3 billion HBV carriers all around the world.The HBV carriers is easily to get the risk of liver fibrosis,liver cirrhosis and liver cancer than non-carriers.China is a high incidence of hepatitis B and have about 120 million HBV carriers.However,the obsolete clinical manifestations and the ignoring of hepatitis B to the majority of patients lead to the aggravate of this disease,even the occuring of chronic hepatitis and liver fibrosis. Chronic hepatitis(HBV-induced) is the caused the important factor of the causing of liver fibrosis.The simple treatment for liver fibrosis can not make it cured completely.even if the well treatment can not rule out the risk of relapse.Therefore, anti-liver fibrosis in chronic hepatitis should be treated as a whole,is the guarantee for the complete cure.The research for the mechanism of chronic hepatitis and liver fibrosis have been some progress all round the world in nowday.However,in the field of clinical treatment,it still can’t achieve the significant progress.Add the efficacy of common drugs for liver fibrosis is beyond ideal,and the drug have some disadvantages,for exsample, the obvious side effects and the high cost.So researchers at home and abroad are trying to search for a drug which have the advantage, including the exact effect,cheap and the shortage of toxic side effects.in order to cure the majority of chronic hepatitis and liver fibrosis.This subject is selecting the JIAWEISANRENTANG as an experimental drug,used for chronic hepatitis and liver fibrosis research.The research adopted immunohistochemist-ry,cell culture,flow cytometry and RealTime-PCR technology for the teatment effect and mechanism of anti-chronic hepatitis and anti-hepatic fibrosis on DHBV model,autoimmune liver fibrosis in rats,HSC cells and 2.2.15 cells.It is supply the clinical and laboratory basis for the further development and application.1.Use the DHBV-positive subjects to research JIAWEISANRENTANG in their serum DHBV-DNA levels.The results showed that: the model group of duck serum DHBV-DNA levels,compared with the normal group were significantly different(P＜0.01), JIAWEISANRENTANG extract high-dose,low-dose and high dose of alcohol extract of DHBV-DNA were significantly inhibited(P＜0.05 or P＜0.01),and JIAWEISANRENTANG small doses of alcohol did not extract a significant role in inhibiting DHBV-DNA.2.Used JIAWEISANRENTANG in the role of the 2.2.15 cells were observed on the 2.2.15 cells and HbsAg secretion HbeAg.The results showed that concentration of 1,0.5,0.25,0.125,0.625,0.3125 mg /mL of JIAWEISANRENTANG tothe secretion of HbsAg and HbeAg for 2.2.15 cell inhibition rate was 85.36%,79.72%,75.81%,69.65%, 62.14%,30.56%and 90.17%,83.26%,76.34%,46.86%,20.32%,0.36%. Drug dose and HbsAg and HbeAg show increased secretion of the relationship between inhibition rate,which increased with the dose of which drug to more pronounced inhibition of HbsAg.3.Observation JIAWEISANRENTANG on 2.2.15 cells,HBV-DNA expression.The results showed that with a flavored soup Sanren 2.2.15 cells inhibited the expression of the role of HBV,of which 1mg/mL,0.5mg/mL and 0.25mg/mL concentration JIAWEISANRENTANG extract more than the positive effect of the drug.4.Rat model of liver fibrosis by DMN was observed for the action to liver liver tissue HA,LN,IVC,PCIINP and the impact of HyP by JIAWEISANRENTANG.Model group and treatment group in serum HA, LN,IVC,PCIII and water HyP average increase compared with the normal group,the difference was significant(P＜0.01). JIAWEISANRENTANG to large and small dose group compared with the model group,serum HA,PCIII,LN,CIV and HyP were significantly decreased(P＜0.05 or 0.01),and the effect increased with the dose corresponding enhancement;JIAWEISANRENTANG alcohol extract of high-dose group compared with the model group,serum LN,IVC, PCIII and HyP level dropped to a certain extent,but no significant (P＞0.05).JIAWEISAN-RENTANG low-dose group compared with the model group,serum PCIII,CIV,HyP levels decline,but no significant(P＞0.05).Fufangbiejiaruanganpian serum HA,PCIII, CIV level than the model group was no significant difference(P＞0.05).5.Observed the rat model of liver fibrosis which is induced by DMN insect JIAWEISANRENTANG on DMN hepatic fibrosis of rat liver tissue the effects of morphology:(1) HE staining;(2)α-SMA immunohistochemistry;(3) I type collagen immunohistochemistry. The experiments show that damage from hepatic loble,the formation of false lobules,hyperplasia of collagen fibers,such as objective changes in liver structure,the JIAWEISANRENTANG can reduce the extent of liver fibrosis and improve liver structure, the promotion of ECM degradation and thus the treatment of experimental liver fibrosis.6.Observed JIAWEISANRENTANG Decoction on rat liver tissue TGFβ1 and PDGF-B Inhibition of the expression.The results showed that the model group and treatment group in serum TGF-β1 and PDGF expression,and the normal group,the difference was significant (P＜0.01).JIAWEISANRENTANG extract group were significantly inhibited TGF-β1 and PDGF expression(P＜0.05),and superior to the positive drug,especially JIAWEISANREN-TANG mention the most obvious high-dose group(P＜0.01),and dose effect with a corresponding increase in enhancement;JIAWEISANRENTANG ethanolic dose low-dose group compared with the model group,the TGF-β1 and PDGF inhibit the effect of the expression of significant (P＜0.05).M JIAWEISANRENTANG decoction group compared with the model group,serum TGF-β1 on the impact of no significant(P＜0.05).7.Observed JIAWEISANRENTANG Decoction on liver fibrosis serum TNF,IL-1 and the effects of IL-6.The results suggest that JIAWEISANRENTANG extract Sanren group have significantly lower serum TNF-α,IL-1,as well as the role of content(P＜0.05 or P＜0.01).Water extract of which high-dose group was superior to the positive effect of the drug group.The JIAWEISANRENTANG high-dose group in the serum TNF-αand IL-1 significantly reduced the role of IL-6 but no significant effect.While small doses of alcohol extracts of serum TNF-α,IL-1 and IL-6 had no significant effect.8.Discussion JIAWEISANRENTANG on HSC-T6 TIMP-1 and MMP-2 expression.The results showed that the concentration of 40mg/mL and 20mg/mL JIAWEISANRENTANG can effectively reduce the TIMP-1 and MMP-2mRNA level and decreased with lower concentrations.9.Observed of rat liver tissue and HSC in the Fas/FasL expression and flavored soup Sanren intervention role.Experiments found that JIAWEISANRENTANG significantly increase rat liver tissue and HSC Fas/FasL gene mRNA expression,and the water extract of the Fas /FasL are better than the increase in the role of drug-positive group. 10.Discussion on HSC apoptosis in vitro and JIAWEISANRENTANG intervention role.Experimental Tip:JIAWEISANRENTANG and Danshen injection can induce apoptosis of HSC,which JIAWEISANRENTANG extract 40mg/ml and 20mg/ml obvious apoptosis dose group,and better than Danshen injection group.In addition, the JIAWEISANRENTANG Decoction group to promote apoptosis of HSC is also a dose-dependent manner.11.Explore JIAWEISANRENTANG Decoction on liver fibrosis in rat tissue plasminogen activator inhibitor,tissue-type plasminogen activator impact.The results showed that:compared with the cells in the control group,JIAWEISANRENTANG adjustable uPA and PAI-1 expression.Which the concentration of 40mg/mL and 20mg/mL JIAWEISANRENTANG extracts of HSC-T6 cells,uPA and PAI-1 expression and regulation of better than other groups.In serum uPA and PAI-1 expression inhibition in JIAWEISANRENTANG group are better than other groups.更多还原