【作者】 李骢；

【导师】 贾玉杰；

【作者基本信息】 大连医科大学 ， 病理学与病理生理学， 2007， 博士

【摘要】 背景和目的肝纤维化是肝脏受到慢性损伤时的一个愈合修复过程,又是所有慢性肝病进展成肝硬化的共同病理基础。有效的治疗肝纤维化可以防止各种慢性肝病向肝硬化发展。近年来,国内外在对肝纤维化启动和进展机制的研究方面取得了一定进展。但是在肝纤维化临床治疗领域,并没有取得明显的进步。许多西药由于抗纤维化作用靶位单一,疗效并不理想,而有些药物毒副作用大于治疗作用,不宜用于临床。我国的中药复方制剂具有多种药效成分协同作用,安全性强的特点,具有显著的优势。中药肝复康是本研究室多年来通过反复实验总结出的抗纤维化经验方,即往的动物实验已多次成功证实了肝复康对肝纤维化组织具有预防及治疗作用。本实验拟在肝星状细胞(hepatic stellate cells,HSC)培养体系中加入血小板衍生生长因子(platelet-derived growth factor,PDGF)及中药血清,应用半定量RT-PCR技术,分别测定肝星状细胞经外源性PDGF处理,再通过不同浓度含药血清干预后,其Ⅰ、Ⅲ型胶原及α-SMA mRNA的变化特点;并以Western blot检测不同方式处理后肝星状细胞STAT、ERK、PI3-K蛋白的不同表达情况。从而进一步揭示中药肝复康对肝星状细胞在肝纤维化中的分子调节机制。在肝纤维化发病的分子机理方面,尽管目前国内外已从基因和转录水平进行了许多成功的研究。然而基因的功能活性主要靠蛋白质来实现,若能直接从蛋白质整体水平入手来研究将更加全面、真实地揭示肝纤维化的演变过程。但迄今为止,有关中药对肝星状细胞的蛋白质组的干预实验尚无报道。本实验将首次开展中药对肝星状细胞和肝组织蛋白质组的干预实验,可望从蛋白质组学角度进一步揭示肝纤维化的发病机制和中药的药理作用,为新药研制提供理论依据。方法一、用RT-PCR方法检测Ⅰ、Ⅲ型胶原及α-SMA mRNA的变化将HSC分为5组:即空白对照组:加10%对照血清的DMEM;模型对照组:加10%对照血清+血小板衍生生长因子(终浓度60ng/ml)的DMEM;肝复康低剂量组:加10%低剂量药物血清+血小板衍生生长因子(终浓度60ng/ml)的DMEM;肝复康中剂量组:加10%中剂量药物血清+血小板衍生生长因子(终浓度60ng/ml)的DMEM;肝复康高剂量组:加10%高剂量药物血清+血小板衍生生长因子(终浓度60ng/ml)的DMEM,分别测定肝星状细胞经外源性PDGF处理,再通过不同浓度含药血清干预后,其Ⅰ、Ⅲ型胶原及α-SMA mRNA的变化特点。二、Western blot检测不同方式处理后STAT、ERK、PI3-K蛋白的不同表达将HSC随机分为6组,分别为:对照组:加10%正常对照血清的DMEM;模型组:加10%正常对照血清的DMEM;中药组:加10%中剂量药物血清;ERK阻断剂组:加U0126(ERK阻断剂,终浓度为10μmol/L);PI3K阻断剂组:加LY-294002(PI3K阻断剂,终浓度为10μmol/L);STAT阻断剂组:加Parthenolide(JAK-STAT阻断剂,终浓度为50μmol/L)。以上各组均作用30分钟后,除对照组外,其余各组均加入血小板衍生生长因子(终浓度60ng/ml)。反应10分钟后,提取不同分组HSC总蛋白样品,行Western blot检测STAT、ERK、PI3-K蛋白的不同表达。三、二维电泳技术检测肝组织及肝星状细胞蛋白质分子的动态表达肝星状细胞2-D电泳:制备正常大鼠肝星状细胞、加入血小板衍生生长因子的肝星状细胞及加入肝复康药物血清和血小板衍生生长因子的肝星状细胞,分别设定为正常对照组、模型组及药物组。分别提取以上各组的总蛋白质并进行等电聚焦电泳,随后进行SDS-PAGE电泳。凝胶染色后比对蛋白质斑点,找出上调和下调的蛋白质。肝脏组织2-D电泳:将对照组、模型组及药物组大鼠肝脏取出,提取组织总蛋白质,分别进行等电聚焦电泳,随后进行SDS-PAGE电泳。凝胶染色后比对蛋白质斑点,找出上调和下调的蛋白质。统计学处理多组比较用方差分析,两组间比较用t检验。结果一、药物血清对肝星状细胞Ⅰ、Ⅲ型胶原mRNA表达的影响在空白对照组,HSCⅠ、Ⅲ型胶原mRNA表达呈相对低水平;加入对照血清及PDGF刺激的模型对照组,HSC细胞显示出对外源性PDGF刺激因子的敏感反应性,与空白对照组相比Ⅰ、Ⅲ型胶原mRNA表达明显上调(P<0.01﹚;在药物血清观测组,与模型对照组相比,由PDGF刺激诱导的HSCⅠ、Ⅲ型胶原mRNA表达增加明显受到抑制(P<0.01﹚;不同药物剂量产生的干预效果不同,肝复康各剂量之间比较,以中剂量组Ⅰ型胶原mRNA表达降低最明显,优于低剂量组(P<0.01﹚,和高剂量组相比无显著性差异;中剂量组Ⅲ型胶原mRNA表达降低亦最明显,但三个剂量组之间相比无显著性差异。二、药物血清对肝星状细胞α-SMA mRNA表达的影响在空白对照组,HSCα-SMA mRNA表达呈相对低水平;而模型对照组α-SMA mRNA表达呈明显升高(P<0.01﹚;药物血清各组则均具有能明显抑制这种升高的作用(P<0.01﹚,其中以中剂量组干预效果最为明显,但三个剂量组之间相比无显著性差异。三、肝星状细胞STAT、ERK、PI3K蛋白的表达情况肝星状细胞STAT、ERK、PI3K蛋白表达在不同干预条件下存在差别,表现为对照组、模型组、中药组、阻断剂组各免疫印迹条带的灰度及面积不同。中药可有效抑制STAT、ERK、PI3K蛋白的表达。此外,在分别应用STAT、ERK、PI3K阻断剂后,除相对应的蛋白质表达明显下调外,其余两种蛋白质的表达也有不同程度的下降。四、HSC蛋白质的差异展示(一)培养12小时后蛋白质的差异展示HSC培养12小时后,分别提取对照组、模型组及中药治疗组HSC的总蛋白质。通过处理后得出:对照组蛋白质斑点数181个,模型组蛋白质斑点数161个,中药治疗组蛋白质斑点数124个。(二)培养24小时后蛋白质的差异展示HSC培养24小时后,分别提取对照组、模型组及中药治疗组HSC的总蛋白质。通过处理后得出:对照组蛋白质斑点数189个,模型组蛋白质斑点数229个,中药治疗组蛋白质斑点数397个。(三)培养48小时后蛋白质的差异展示HSC培养48小时后,分别提取对照组、模型组及中药治疗组HSC的总蛋白质。通过处理后得出:对照组蛋白质斑点数118个,模型组蛋白质斑点数183个,中药治疗组蛋白质斑点数81个。(四)不同治疗时间蛋白质的差异展示随着治疗时间的不断延长,HSC的蛋白质表达在不同时间有了不同变化.有的变化发生在治疗后24小时,有的则发生在治疗后的48小时。五、肝组织蛋白质的差异展示造模4周后,模型组与治疗组肝组织总蛋白质存在着一定差异。具体体现为:蛋白质斑点数不同,模型组217;治疗组241。同时,部分蛋白质在两组中表现出不同的丰度。结论一、经肝复康含药血清处理,可下调HSC-T6细胞株Ⅰ、Ⅲ型胶原、α-SMA mRNA表达,不同浓度含药血清作用强度也有所不同。其中以中剂量组干预效果最为明显。中药肝复康对肝纤维化具有明显的抑制作用,其作用机制可能是通过非特异性干扰PDGF功能,抑制Ⅰ、Ⅲ型胶原、α-SMA mRNA的转录,以达到抗纤维化的作用。二、应用中药肝复康可通过干预HSC主要信号转导途径,从而减弱STAT、ERK、PI3-K的表达,以达到抗肝纤维化的目的。同时,在分别应用STAT、ERK、PI3K阻断剂后,除相对应的蛋白质表达明显下调外,其余两种蛋白质的表达也有不同程度的下降,因此,我们猜测在肝星状细胞内Ras/ERK、PI3K和JAK/STAT途径之间存在着相互作用,既cross-talk。三、在对三个不同时间段的模型组肝星状细胞蛋白质的凝胶图谱比较发现:每一时间段都有与肝纤维化相关的蛋白质表达,并且随着时间的不断延长,蛋白质的数量也有所变化。这些现象提示了肝纤维化的发生与多种蛋白质的作用有关。四、中药肝复康治疗肝纤维化是通过调节多个蛋白质表达产生的。随着中药治疗时间的不断延长,肝星状细胞中药治疗组的蛋白质斑点也有了一定的变化。体现出中药复方多成分、多环节、多层次、多靶点的综合药理作用。更多还原

【Abstract】 Background and objective:Liver fibrosis is characterized by an accumulated deposition of extracellular matrix materials (ECMs) and the resulting reconstruction of liver parenchyma, companying the impairment of normal liver function. A clinical and experimental study has found that liver cells, hepatic stellate cells (HSC), kupffer cells and sinus endothelial cells all take part in the formation of hepatic fibrosis, in which HSC plays a very important role. Activation of HSC is commonly regarded as the major link of hepatic fibrosis and the main resource of synthesis of ECM. The main characteristic of activation of HSC is excessive proliferation of HSC. In addition,α-SMA is regarded as a marker of activation of HSC Activation of HSC can lead to excessive synthesis of collagen. HSC proliferation is stimulated by growth factors derived from hepatocytes, Kupffer cells, infiltrating macrophages, platelets, and activated HSCs themselves in paracrine and autocrine manners. In particular, PDGF(platelet-derived growth factor,PDGF), the most potent mitogen for HSCs, stimulates their DNA synthesis by activating extracellular signal-regulated kinas(ERK), phophatidylinositol 3-kinase (PI3K) and signal transducer and activator of transcription(STAT) pathways. Hence, PDGF play a very important role in proliferation, activation of HSC and synthesis of ECM. Traditional Chinese medicine has shown its own advantage in treating some difficult diseases. Developed by our department compound Ganfukang has been used as the traditional Chinese medicine for treating hepatic fibrosis. Clinical observations in our affiliated hospital have proved its effective. However, further study of its detailed anti-hepatic fibrosis mechanism is still needed. On the basis of theabove mentioned theory and research developments, our study with the cell culture as technical platform, was to observe the influence of serum collected from rats perfused with Ganfukang on activation and proliferation of HSC in vitro, and using transcription-polymerase chain reaction (RT-PCR) and image analysis technology to observe its influence on the expressions ofα-SMA, collagens I, III, using western blotting to observe its influence on the expressions of protein ERK, PI3K and STAT, particularly through disturbing platelet-derived growth factor dependent signaling pathways and cross-talk among these pathways.With the rapid development of proteomic methods, it is possible to find and identify the direct sites of Chinese medicine by using comparative proteomic methods. Two-dimensional gel electrophoresis (2-DE) is currently the most common analytical technique available for the study of protein expression patterns. Our study is to observe the influence of Chinese medicine on liver tissues and HSC by using 2-DE. In this way, we can supply the theory basement for drug development.Materials and methods:1 Cell culture and grouping for RT-PCRAccording to intervening factors, HSCs were divided into 5 groups, i.e. control group, model group, high dosage serum group, medium dosage serum group and low dosage serum group. Upper culture medium was removed after cultivated for 24 h. Control group serum:10% normal SD rat serum +DMEM, model group serum: 10% normal SD rat serum +DMEM+PDGF(final concentration 60ng/ml), high dosage group serum: 10% high dosage serum +DMEM+PDGF(final concentration 60ng/ml), medium dosage group serum: 10% medium dosage serum + DMEM + PDGF(final concentration 60ng/ml) and low dosage group serum: 10% low dosage serum +DMEM+PDGF(final concentration 60ng/ml) were accordingly added. Total RNA was extracted from HSCs. Expression of mRNA was determined by reverse transcription polymerase chain reaction2 Cell culture and grouping for western blottingHSC cells were randomly assorted for six groups, those were control group(10% normal SD rat serum +DMEM), model group(10% normal SD rat serum +DMEM), Chinese herb-treated (with 10%medium dosage serum )group, ERK blocker-treated (U0126, final concentration 10μmol/L)group, PI3K blocker-treated (LY-294002, final concentration 10μmol/L )group and STAT blocker-treated (Parthenolide, final concentration 50μmol/L )group, After the treatments of PDGF (10% normal SD rat serum +DMEM+PDGF:final concentration 60ng/ml),and Ganfukang (10% medium dosage serum + DMEM + PDGF:final concentration 60ng/ml) at defined concentration and time(12h, 24h,48h respectively). Cells of control, PDGF-treated or drug-treated, were harvested. Immunoblotting was used to determine the expressions of STAT, ERK and PI3K.3 Two-dimensional gel electrophoresisHSCs were assorted for three groups, those were control group, model group, Chinese herb-treated group; Live tissues were divided into 3 groups. Those were control group, model group and Chinese herb-treated group. All protein from those groups was extracted. The method of O’Farrell, with modification, was followed . For the first-dimension, isoelectric focusing was performed by using a vertical mini-IEF system (Jim-X Company) and using analytical SDS-PAGE carried out the second-dimension separation.Statistical analysisValues reported in the figures represent means±SD of three or more independent samples. The results were analyzed by the unpaired Student’s t-test. Statistical significance was set at P<0.05.Results1 Effect of each group serum on collagen I, III mRNA expression of HSC in ratsBy using RT-PCR, we investigated effects of Ganfukang serum on collagen I, III mRNA expression. It shows that Ganfukang serum decreases collagen I, III mRNA expression of HSC in a concentration-dependent manner. PDGF can increase the expression of collagen I, III mRNA obviously compared with control group P<0.01. Ganfukang serum can decrease collagen I, III mRNA expression significantly compared with model group p<0.01,especially medium dosage serum has the most effective function on inhibition the expression of collagen I mRNA, which has the significant difference compared with low dosage group P<0.01,while there is no significant difference between high dosage group and medium dosage group.2 Effect of each group serum onα-SMA mRNA expression of HSC in ratsRT-PCR shows that Ganfukang serum decreasesα-SMA mRNA expression of HSC in a concentration-dependent manner. PDGF can increase the expression ofα-SMA mRNA obviously compared with control group P<0.01. Ganfukang serum can decreaseα-SMA mRNA expression significantly compared with model group p<0.01, especially medium dosage serum has the most effective function on inhibition the expression ofα-SMA mRNA. While there is no significant difference among these three different Ganfukang dosage groups.3 Protein expression of STAT, ERK and PI3K assay by western blottingWestern blots show the effects of Ganfukang, ERK blocker, PI3K blocker, STAT blocker on PDGF-induced STAT, ERK and PI3K protein levels. The protein levels increased by PDGF. However, The protein levels stimulated by PDGF were significantly inhibited not only by Ganfukang or it’s own blocker, but also by the other two blockers.4 Comparison of the protein patterns of HSCsThe HSCs were harvested after 12-hour,24-hour and 48-hour treatments of PDGF and Ganfukang respectively. The proteins of control group, PDGF -treated group, and Ganfukang-treated group were extracted and prepared for comparative proteomic analysis. Within the applied pH range (PH 4.0~ 6.0), a protein load of 30μg per 8 cm strip was the optimal quantity for silver-stained gels. Gel scans were analyzed for spot numbers using PDQuest software. After treatments 12 hours, there were 188 spots that the gel of control group contained , 161 spots were found on the gel of PDGF-treated group, the gel of Ganfukang-treated group contained 124 spots. After treatments 24 hours, there were 189 spots that the gel of control group contained,229 spots were found on the gel of PDGF-treated group, the gel of Ganfukang-treated group contained 397 spots. After treatments 48 hours, there were 118 spots that the gel of control group contained,183 spots were found on the gel of PDGF-treated group, the gel of Ganfukang-treated group contained 81 spots. There are many kinds of alterations on protein spots in different time of treatment.5 Comparison of the protein patterns of liver tissuesFor the liver tissues,there were 217 spots that the gel of model groupcontained, the gel of Ganfukang-treated group contained 241 spots.Conclusions:1 The anti-fibrosis roles of Ganfukang compound maybe influence the function of PDGF by nonspecific action, thereby inhibit the transcription of I、III collagen、α-SMA mRNA and decrease the production of ECM.2 The activity of ERK can be inhibited by its inhibitor, while the activity of PI3K and STAT can also be inhibited by ERK inhibitor. So there maybe exist crosstalk among these three pathways. Such crosstalk may contribute to development of liver fibrosis.3 It was found that there are different protein spots in every different phase by comparing Gel scan among three model groups. The number of protein is changed as the time prolonged. This shows that the pathogenesis of liver fibrosis is involved with many kinds of protein.4 There is a significant difference at protein level between untreated and PDGF-, Ganfukang- treated cells. It suggests that the differential expression analysis of proteomes may be useful to further study on mechanism of Ganfukang, and search for new targets of anti-liver fibrosis medicine.更多还原