【作者】 罗大有；

【导师】 张奉学；

【作者基本信息】 广州中医药大学 ， 中西医结合基础， 2011， 博士

【摘要】 肝纤维化是肝细胞受到炎症刺激及发生坏死时,肝脏内纤维结缔组织异常增生的一种慢性、渐进性的病理过程,是慢性肝病向肝硬化发展的必经环节。有资料表明,肝硬化病人已占我国内科总住院人数的4.3%-14.2%,其病死亡率在消化系统疾病中占居第二位,仅次于恶性肿瘤。肝纤维化是一种慢性、进行性、弥漫性的肝脏病变,各种肝脏病变长期反复发作均可以导致肝纤维化的生成。因此,能否延缓、阻断或逆转肝纤维化的发展,具有重大的研究和应用意义。健脾软肝方为现代组方,是根据传统中医理论“肝病实脾”的理论并结合科学辩证而生成的。健脾软肝方的组方中包含了太子参、黄芪、白术、土鳖虫、枳壳、甘草、等多种中药,本组方中的各单味药分别具有补脾益气燥湿,和中缓急,扶正化邪、活血化瘀、通络理气,柔肝调肝等作用。虽然健脾软肝方在临床上得到广泛的应用,但对健脾软肝方抗肝纤维化机理的研究相对较少。因此,以此作为本实验的研究基点。本研究首先通过确定健脾软肝方对四氯化碳大鼠肝纤维化模型的治疗作用,通过测定各药物组的肝指数,用赖氏法检测各组间ALT、AST的水平以及HE染色法评价各药物组和模型组间的肝纤维化程度。随后在同一模型中,检测健脾软肝方对大鼠肝纤维化模型中TGF-β/Smad通路、经典WNT通路和PI3K通路中相关因子的表达影响。在TGF-β/Smad通路的研究中,利用RT-PCR法检测健脾软肝方各剂量组中大鼠TGF-β1基因的表达水平,利用免疫组化法检测健脾软肝方各剂量组对TGF-β受体数目的影响,以及用western blot法检测各实验组中肝组织内SMAD3和SMAD7蛋白的表达。而在经典WNT通路的研究中,以PCR手段检测所有相关基因的表达,包括了WNT-3a蛋白、FRZ1和β-catenin,同时也检测了HNF-4和HNF-6基因的表达水平。在PI3K通路的研究中,主要是利用了RT-PCR法检测了PDGF和PI3K mRNA的表达水平,放免法检测各剂量组中大鼠血清的cAMP水平,并利用western blot法检测TIMP-1和MMP-3蛋白的表达水平。此后,再进一步研究脾脏功能的变化对肝纤维化的影响。利用四氯化碳大鼠肝纤维化脾次切除模型,用流式细胞仪来检测各药物组大鼠全血中CD4和CD8细胞亚群比例的变化,以及免疫扩散法检测大鼠血清中IgG的分泌水平。最后通过体外实验,在测定健脾软肝方在大鼠星状细胞HSC-T6细胞的半数毒性浓度。其TC50为0.128mg/ml,以此为基础分别设定三个剂量,分别是TC500. 1mg/ml, TC10 0.025mg/ml和TC0 0.00625mg/ml为实验药物浓度。用流式细胞仪以观察健脾软肝方对大鼠HSC细胞上TRAIL凋亡受体的影响。在本研究中,利用大鼠肝纤维化模型和体外HSC-T6星状细胞模型相结合,多种检测手段并从多个方面对健脾软肝方的机制进行阐述。通过四氯化碳大鼠肝纤维化模型确定健脾软肝方对肝纤维化的治疗作用。在实验中发现,与模型组相比,健脾软肝方高剂量110g·kg-1组和中剂量40g·kg-1组均有较好的抗肝纤维化作用。表现在能够明显降低肝纤维化大鼠的肝指数,降低ALT和AST水平并且缓解肝纤维化进程。而低剂量组20g·kg-1组对四氯化碳大鼠肝纤维化的治疗作用相对较弱。随后利用四氯化碳大鼠肝纤维化模型,从多个肝纤维化细胞／因子通路对健脾软肝方的机制进行研究。发现健脾软肝方对肝纤维化的TGF-β/Smad通路、经典WNT通路和PI3K通路均有调节作用。在TGF-β/Smad通路的研究中发现,健脾软肝方高剂量组、低剂量和中剂量组对肝纤维化大鼠中的TGF-β1的mRNA有明显的抑制作用,而且能显著下调TGF-β受体数目,并且能够上调SMAD3、下调SMAD7蛋白的表达,调节SMAD3/SMAD7比例。模型组中的TGF-β1受体的阳性比例达到了43%,而健脾软肝方高剂量组和中剂量组中均少于10%的阳性率,明显低于模型组。而SMAD3/SMAD7蛋白表达比例在模型组为2.01,而健脾软肝方高剂量组和中剂量组分别1.08、1.32,低剂量组则为1.63,显示低剂量组对调节SMAD蛋白的作用稍弱。在经典WNT通路的相关基因的表达中发现,健脾软肝方各剂量组对WNT-3α的表达均有抑制作用,但主要还是通过上调FRZ受体来达到肝纤维化的作用。同时,健脾软肝方还能直接抑制β-catenin的表达。此外,健脾软肝方高剂量组和中剂量组对HNF-4和HNF-6都具有诱导其表达的作用,与模型组相比,表达明显增强。在肝纤维化PI3K通路的研究中,主要以PDGF、PI3K基因的表达和TIMP-1、MMP-3蛋白的表达为切入点。发现健脾软肝方能够明显抑制PDGF、PI3K基因的转录水平,提高机体cAMP合成水平,并能调节TIMP-1和MMP-3的比例。在模型组中,肝纤维化的cAMP水平为47.69fmol/μl,高剂量组、中剂量组和低剂量组分别为86.57、76.87和69.88fmol/μl。在本研究中,也结合了中医理论“肝病实脾”,结合了大鼠肝纤维化中脾脏功能的异常是否与肝纤维化的进程相关。初步研究证实,在大鼠的肝纤维化模型中,大鼠的脾脏功能会出现紊乱,主要表现在CD细胞亚群的失调,IgG过度增高。显示在肝纤维化进程中,机体的自身的过度免疫或者脾脏功能的紊乱都对肝纤维化有加重作用。在该研究中,利用了四氯化碳大鼠肝纤维化脾次切除模型作为研究对象。发现在肝纤维化进程中,对大鼠脾脏进行次切除术能够明显降低大鼠过度的自身免疫,并且能够调节CD细胞亚群的比例。而健脾软肝方高剂量组和中剂量组均能有效调节机体的免疫能力,防止机体过度自身免疫所造成的肝损伤。其中模型组中的CD4/CD8仅有0.85,而脾次切除组为2.21,健脾软肝方高剂量组、中剂量组和低剂量组分别为2.36、2.25和1.82。最后在体外HSC-T6细胞系的研究中,利用流式细胞仪发现健脾软肝方在0.1mg/ml对细胞上TRAIL凋亡受体的表达有明显的诱导作用。与正常HSC-T6细胞组TRAIL的阳性率23.88%相比,健脾软肝方0.1mg/ml组与0.025mg/ml组的阳性率分别为39.14%、32.72%,分别升高15.26%和8.84%。综合上述,健脾软肝方可以通过多种渠道发挥抗肝纤维化的作用。健脾软肝方可以影响多种细胞信号通道的,可以从TGF-β/Smad通路、经典WNT通路和PI3K通路来抑制肝纤维化的进展。同时对机体的免疫功能具有调节作用,可以提高CD4/CD8细胞亚群的比例,并能减弱在肝纤维化过程中机体自身过度的免疫反应对肝脏造成的损伤。同时在凋亡方面,健脾软肝方可以直接上调活化HSC-T6细胞上的TRAIL凋亡受体,使活化的HSC-T6细胞进入程序性死亡阶段而发挥抗肝纤维化的作用。更多还原

【Abstract】 Hepatic Fibrosis is a chronic disease caused by the injuries of hepatic cells. It will lead to the cirrhosis, and even dead. According to the data from clinical, the percentage of cirrhosis patients in the hospital all over the country is about 14.2%, which the mortality takes the second place in the digestive system diseases. So it is worth to study how to deferred and reverse the development of hepatic fibrosis.The prescription of Jianpiruangan is a modern prescription according to the tra-ditional Chinese theory of "therapy the hepatic with spleen" combined with the di-fferentiation of symptoms and signs. In this prescription, it contains varies herbs, such as Radix Pseudostellariae, Radix Astragali, Atractylis ovata, tuckahoe, Chinese angelica root. White Paeony Root, Danshen Root, Ground Beeltle, oyster, Pangolin Scales, Bitter Orange, liquorice, hawthorn and so on. Every single herb has its own functions in the anti-hepatic fibrosis. This prescription is well used in the clinical, but the mechanism is seldom described or was studied. So we will take a deep research of this prescription to fine out how this prescription takes effects on the anti-hepatic fi-brosis.We studied this prescription in vivo an in vitro. In vitro experiments, we confirmed the therapeutic effects of this prescription and found that it could reduce the hepatic index in the hepatic fibrosis rats model induction by carbon tetrachloride, lowered the levels of ALT and AST, relieved the degrees of hepatic fibrosis. And in the next in vitro experiments, we found that this prescription could affect multifold cellular signal tunnels in the hepatic fibrosis. In the research of TGF-β/Smad tunnel, we found that this prescription could reduce the expression level of TGF-βand the numer of the TGF-βreceptors in the cells, and adjusted the proportion of the SMAD3 and SMAD7. In the research of classical WNT tunnel, we found that this prescription took effects on the anti-hepatic fibrosis was to enhanced the expression level of FRZ, but not the expressions level of WNT protein. And this prescription could reduced the expression level ofβ-catenin, but enhanced the expression of HNF-4 and HNF-6. In the research of PI3K tunnel, this prescription coudld reduce the transcription of PDGF and PI3K mRNA, raised the cAMP level in the serum, and adjusted the proportion of TIMMP-1 and MMP-3 proteins. And in the further studies, we found the rats with hepatic fibrosis got the functional turbulence in the immune system, which showed the lower proportion of the CD4/CD8 by the rats hepatic fibrosis model with spleen subordinated Resectionlie. The prescription of Jianpiruangan could reduce the secretion of IgG, and adjusted the proportion of CD subcellulars. At last, in the vivo experimental research, we found that the concentration of this prescription of 0.128mg/ml and TC10 0.025mg/ml, could enhance the number of the TRAIL receptors directly which lead the HSC-T6 cells to the apoptosis.According to the above studies, we knew that the prescription of Jianpiruangan has a good effect on the anti-hepatic fibrosis. It could affect multifold sides of the development of hepatic fibrosis, such as the cellular tunnel, adjustment the function of immune system and induction the cells to apoptosis.更多还原