【作者】 解克芳；

【导师】 王继峰；

【作者基本信息】 北京中医药大学 ， 中西结合基础， 2008， 博士

【摘要】 研究背景及目的肺纤维化是一种病因复杂的疾病,以肺成纤维细胞过度增殖、细胞外基质过度沉积为主要特征。其发病机理被认为与各种细胞因子、生长因子之间的平衡失调有关。在肺纤维研究中,细胞因子网络始终是人们的研究热点,其中转化生长因子-β(transforming growth factor-β,TGF-β)被认为是导致纤维化的重要因子。结缔组织生长因子(connective tissue growth factor,CTGF)作为TGF-β的下游因子,可能是肺纤维化过程中的一个关键的调节因子。CTGF是即刻早期基因CCN (CTGF, cyr61, nov)家族成员之一。为富含半胱氨酸的多肽。在TGF-β诱导下,可由多种细胞分泌合成,它也具有促细胞增殖和细胞外基质(extracellular matrix, ECM)合成等作用,因此CTGF已成为这一研究领域中新的热点。至今,CTGF在肺纤维化中的作用知之甚少,本论文即探讨CTGF在肺成纤维细胞中的生成与ERK1/2途径的关系。姜黄素(Curcumin )是从姜黄中提取的一种活性成分,现代药理研究认为姜黄素具有抗炎、抗凝、降脂、抗氧化和抗肝纤维化等多种作用。为此我们推测,姜黄素对肺纤维化也具有预防和治疗的作用。前期的整体实验研究已经证明了这一点。研究方法及结果1文中第一部分,我们探讨了姜黄素对体外培养人胚肺成纤维细胞(MRC-5)增殖、胶原表达和周期分布的影响,方法:体外培养人胚肺成纤维细胞(MRC-5),用姜黄素处理,分为①正常对照组,②10μmol/L姜黄素组,③20μmol/L姜黄素组,④30μmol/L姜黄素组,⑤40μmol/L姜黄素组,⑥50μmol/L姜黄素组,⑦60μmol/L姜黄素组。在37℃继续培养24 h后,用四甲基偶氮唑盐(MTT)法检测细胞增殖情况,用天狼猩红染色法测定细胞内胶原的表达,用流式细胞仪检测细胞周期的变化。结果显示,一定浓度姜黄素能抑制人胚肺成纤维细胞的增殖和胶原的增加,并使细胞周期停滞在G1期,提示姜黄素可能通过抑制人胚肺成纤维细胞的增殖来减少细胞外基质积聚,从而发挥其抗肺纤维化的作用。2文中第二部分,我们建立了肺纤维化体外细胞模型.方法::体外培养人胚肺成纤维细胞,分成四组①正常对照组②TGF-β刺激组③CTGF刺激组④TGF-β+CTGF刺激组,Western-blotting检测胶原Ⅰ、胶原Ⅲ及FN的变化,RT-PCR检测α-SMA的表达水平,酶联免疫吸附实验检测培养液中TIMP-1表达量,天狼猩红染色,检测总胶原的表达,MTT检测细胞的增殖。结果显示,TGF-β联合CTGF能显著诱导胶原Ⅰ、胶原Ⅲ及FN的表达,并且能活化成纤维细胞为肌成纤维细胞促进细胞外基质的沉积,由此可知,TGF-β联合CTGF刺激人胚肺成纤维细胞建立的细胞模型能非常全面的反映肺纤维化过程中成纤维细胞的增殖和细胞外基质的沉积。3文中第三部分,我们讨论了TGF-β刺激下CTGF的生成情况及与ERK1/2途径的关系方法:体外培养人胚肺成纤维细胞,不同浓度的TGF-β刺激,Western-blotting检测CTGF的表达,体外培养人胚肺成纤维细胞,10 ng/ml TGF-β分别刺激30min、1h、3h、6h、12h、24h。酶联免疫吸附实验检测各组细胞中的p- ERK1/2和total- ERK1/2,结果显示,(1)TGF-β可剂量依赖性诱导CTGF的生成,(2)TGF-β可激活ERK1/2信号途径,(3)阻断剂组CTGF的表达也下降,4文中第四部分,我们讨论了姜黄素对CTGF的表达和ERK1/2途径的影响。方法:体外培养人胚肺成纤维细胞分成五组,①正常对照组②TGF-β刺激组③阻断剂预处理,TGF-β刺激组④TGF-β+40umol/L姜黄素⑤TGF-β+60umol/L姜黄素,免疫组化和western-blotting检测每组中CTGF的蛋白的表达,RT-PCR检测检测每组中CTGF mRNA的水平,western-blotting检测各组细胞中p- ERK1/2和总- ERK1/2表达水平。结果显示,姜黄素可抑制CTGF的生成,并能抑制和延缓. p- ERK1/2和总- ERK1/2的表达,故推测,ERK1/2途径参与了CTGF的生成,而姜黄素能抑制两者的表达。结论1姜黄素能体外抑制成纤维细胞的增殖,降低细胞外基质的含量,从而发挥预防和治疗肺纤维化的作用。2 TGF-β联合CTGF孵育成纤维细胞建立的肺纤维化体外细胞模型可较好的反映肺纤维化过程中成纤维细胞的活化增殖和细胞外基质沉积的变化。3 TGF-β可部分通过ERK1/2途径诱导CTGF的表达。4姜黄素可能部分通过抑制ERK1/2途径抑制CTGF的表达。更多还原

【Abstract】 Background and ObjectiveThe pathogenesis of pulmonary fibrosis is complicated, it is characterized with myofibroblast hyperplasia and deposition of extracellular matrix.Study unraveling the complexities of the cytokine and growth factor networks that underpin pulmonary fibrosis has always been an important thing in our understanding of these events. In particular, transforming growth factor-β(TGF-β)appears to be a pivotal contributor to pulmonary fibrosis. Many experiments demonstrated that the pathogenesis is associated with disorders of all kinds of cytokines or growth factors.Connective tissue growth factor(CTGF),which is a downstream cytokine of transforming growth factor-β(TGF-β), may be a critical cytokine in the development of bleomycin-induced pulmonary fibrosis . CTGF is a 38 kDa cysteine-rich peptide, which belongs to the CCN (cyr61, CTGF, and nov) family of immediate early response genes. It acts as a downstream mediator of TGF-β1,It also can induce cell proliferation and extracellular matrix (ECM) deposition. To date, litle is known about the functional role of CTGF in the pulmonary fibrosis. In this paper, we discuss the association of CTGF with TGF-β1 and ERK1/2 signaling way in pulmonary fibrosis.Curcumin is one of the effective element extracted from curcuma. It owes various biological functions, including antiflammation, anticoagulation, decreasing lipid, antioxidation, and inhibiting hepatic fibrosis. From the theory above, we postulate that curcumin may do precautionary and therapeutic effect on pulmonary fibrosis. It have been testified from Experimental studies in vivo we have done that curcumin also have inhibiting pulmonary fibrosis.Methods and Results1 In first part,we investigate the effect of curcumin on proliferation inhibition and expression of collagen and the cell cycle distribution of human embro fibroblasts (MRC-5),Method: The human embryo fibroblasts (MRC-5) were allocated into seven groups:①normal group,②10μmol/L curcumin group,③20μmol/Lcurcumin group,④30μmol/L curcumin group,⑤40μmol/L curcumin group,⑥50μmol/L curcumin group;⑦60μmol/L curcumin group;24 hours later, the proliferation activity of human embro fibroblasts was detected by MTT assay,and the expression of collegan was observed by sirius red staining method, the cell cycle phase was analyzed by flow cytometry. The results indicated that curcumin can suppress the proliferation activity of human embryo fibroblasts and the expression of collagen, It also made cell cycles arrested at G0/G1 phase which implies curcumin may resist the pulmonary fibrosis by suppressing the proliferation activity of human embro fibroblasts and decreasing the expression of collagen.2 In the second part,we established a cellular model of pulmonary fibrosis in vitro.Method:Cultured HLF were divided into four groups①control groups②TGF-β1groups③CTGF groups④TGF-β1+CTGFgroups; Western Blotting were performed to detect the levels of colⅠ、colⅢand FN; RT-PCR were performed to detect the levels ofα-SMA, ELISA were performed to detect the levels of TIMP-1, sirius red staining were performed to detect the levels of the expression of collegan. And the proliferation activity of human embryo fibroblasts was detected by MTT assay.The result indecated that TGF-β1and CTGFcould significantly reduce the COLⅠ、COLⅢ、and FN formation.It also can actived HLF, promote the transdiferentiation of human embro fibroblasts towards myofibroblast and further promote ECM accumulation..So it can concluded that model of human embryonic lung fibroblast activation stimulated with TGF-βand CTGF can reflect more intact HLF activation effects..3 In the third part,we observed the expression and regulation of CTGF by TGF-β1stimulates, and study the interations betwen ERK1/2 signaling pathway and the expression of CTGF on HLF.Method: (1)Cultured HLF were stimulated by TGF-β1. Western-blotting were performed to detect the levels of CTGF protein after treatment. HLF without stimulation were used as control(2) Cultured HLF were incubated with l0ng/ml of TGF-β1,for 30min、1h、3h、6h、12h、24h,ELISA were performed to detect the levels of p- ERK1/2 and total- ERK1/2Result: (1) TGF-β1up-regulated the protein level of CTGF.(2) ERKI/2 signaling way were activated by TGF-β1,(3) Blockade of ERKI/2 activation reduced the level of CTGF protein.4 In the forth part,we examined the effect of curcumin on the expression of CTGF protein.and the ERKI/2 signaling wayMethod: cultured HLF were divided into five groups:①the control group,②TGF-β1stimulated group③PD98059 pretreated ,then TGF-βstimulated group④TGF-β1and 60umol/L curcumin group⑤TGF-β1and 40umol/Lcurcumin , immunohistochemistry and western-blotting were performed to detect the levels of CTGF protein of each group,RT-PCR were performed to detect the levels of CTGF mRNA.of each group, western-blotting were performed to detect the levels of p- ERK1/2 and总- ERK1/2.The results indicated that curcumin can partly inhibit the expression of CTGF and p- ERK1/2 and total- ERK1/2.Conclusions1 Curcumin can suppress the proliferation activity of human embro fibroblasts and the expression of collagen, thus exertive prevention and the treatment pulmonary fibrosis function2 The cellular model of pulmonary fibrosis in vitro we have established use TGF-β1and CTGF stimulates HLF is well reflect the chang of fibroblasts proliferation and the fomation of ECM.3 CTGF is partly induced by TGF-β1.4 Curcumin can partly suppress the expression of CTGF by ERK1/2 signaling way.更多还原