【作者】 冯海利；

【导师】 杨方； 王献华；

【作者基本信息】 华北煤炭医学院 ， 病理学与病理生理学， 2010， 硕士

【摘要】 肺纤维化(pulmonary fibrosis)是多种原因引起慢性肺疾病的共同结局,其病理特点是肺部炎症反应导致肺组织持续性损伤、修复、组织结构的重建及细胞外基质(包括Ⅰ、Ⅲ型胶原)的过度沉积。近年来研究发现在肺纤维化形成与发展过程中细胞因子所发挥的作用极为重要,这些细胞因子包括转化生成因子-β(transforming growth factor-β,TGF-β)、血小板源性生长因子(platelet derived growth factor,PDGF)、结缔组织生长因子(connective tissue growth factor,CTGF)、胰岛素样生长因子(Insulin-like Growth Factor-1,IGF-1)等等。其中TGF-β是研究较广泛和深入的细胞因子之一。包括TGF-β1、TGF-β2和TGF-β3三种形式,它们的生物学特性基本相同,其中TGF-β1最为重要。TGF-β1能有效刺激成纤维细胞分裂、增殖,刺激细胞外基质(extra cellular matrix,ECM)的合成与分泌。TGF-β信号传导包括依赖Smads蛋白家族的TGF-β信号传导通路和丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)的TGF-β信号转导通路。MAPK信号转导通路主要包括细胞外信号调节激酶1/2(extracellular signal-reg-ulated kinase,ERK1/2)、JNK(C-Jun氨基末端激酶,C-Jun N-terminal kinase,JNK又称SAPK1)、P38(Mitogen-activated Protein Kinase)和ERK5(Extracellular Signal Regulated Kinase5)等转导通路。其中JNK是MAPK主要的通路之一。近年来已有研究证实JNK通路参与心、肝、脑等器官纤维化的发生与发展。N-乙酰基-丝氨酰-天门冬酰-赖氨酰-脯氨酸( N-acetyl-seryl- aspartyl-lysyl-proline,AcSDKP)是一种生理性造血系统生长抑制因子。近年来研究发现AcSDKP能抑制心脏、肾脏间质细胞的增生及其胶原的沉积。在我们前期的实验中已经证实,AcSDKP能够抑制矽肺大鼠肺内TGF-β1因子的表达,减少肺内胶原的沉积,AcSDKP具有抑制矽肺纤维化的作用。然而,AcSDKP是通过TGF-β1介导的哪条信号转导通路的调节而发挥其拮抗矽肺纤维化的作用尚不清楚。本实验将研究、探讨AcSDKP是否通过抑制TGF-β1介导的JNK途径,进而抑制肺成纤维细胞的增殖和胶原合成?为揭示AcSDKP抗矽肺纤维化的作用机制进一步提供实验依据。目的:1.探讨TGF-β1介导的JNK信号转导通路在大鼠肺成纤维细胞增殖及胶原合成中的作用。2.探讨AcSDKP能否通过阻断TGF-β1/JNK信号转导通路,进而抑制大鼠肺成纤维细胞增殖和胶原合成。3.为阐明AcSDKP抑制肺纤维化的作用机制提供一些实验依据。方法:1.新生Wistar大鼠肺成纤维细胞原代及传代培养,第四代细胞用于实验研究。2.采用MTT法检测肺成纤维细胞的增殖情况。3.采用激光共聚焦扫描显微镜观察Phospho-JNK蛋白在细胞内的定位与分布情况。4.采用免疫细胞化学法、Western blot法检测JNK、Phospho-JNK、c-jun以及Ⅰ型和Ⅲ型胶原蛋白在细胞内的表达情况。5.实验分组①0.4%血清组(Contorl):以0.4%血清浓度DMEM作为基础培养液。②TGF-β1刺激组(TGF-β1):0.4%血清培养条件下,给予TGF-β1(5μg /L)孵育细胞40min,观察TGF-β1对肺成纤维细胞增殖和胶原合成的影响。③SP600125干预组(SP600125): 0.4%血清培养条件下,SP600125(10nmol/L)预孵育45min后再给予TGF-β1(5μg/L)共孵育细胞40min,观察SP600125对TGF-β1诱导的JNK信号转导通路和肺成纤维细胞增殖和胶原合成的影响。④AcSDKP干预组(AcSDKP): 0.4%血清培养条件下,AcSDKP(10-8 mol/L)预孵育45min后给予TGF-β1(5μg/L)共孵育细胞40min,观察AcSDKP对TGF-β1/JNK信号转导通路的调节以及对肺成纤维细胞增殖和胶原合成的影响。6.采用单因素方差分析进行组间均数比较,以P<0.05表示差异有显著性。结果:1. MTT法检测结果显示,1×105/ml为大鼠肺成纤维细胞最适生长密度;在0.4%血清培养条件下,TGF-β1在5μg /L的浓度时促细胞增殖作用最强;而SP600125和AcSDKP均可以抑制TGF-β1诱导的肺成纤维细胞的增殖。2.激光扫描共聚焦显微镜结果显示,经TGF-β1刺激后胞浆中Phospho-JNK荧光强度的核浆比值较对照组明显增加,SP600125干预组和AcSDKP干预组胞浆中Phospho-JNK荧光强度的核浆比值较TGF-β1刺激组明显减小。3. Western blot法检测结果显示,TGF-β1刺激组Phospho-JNK、c-jun、Ⅰ型与Ⅲ型胶原蛋白表达较对照组明显增加;SP600125和AcSDKP干预作用后,Phospho-JNK、c-jun、Ⅰ型与Ⅲ型胶原蛋白表达较TGF-β1刺激组明显减少,而JNK蛋白表达无明显改变。结论:TGF-β1能够通过TGF-β1介导的JNK通路刺激肺成纤维细胞增殖和Ⅰ型、Ⅲ型胶原蛋白的表达,而AcSDKP能够通过阻断TGF-β1诱导的JNK通路进而抑制大鼠肺成纤维细胞增殖和Ⅰ型、Ⅲ型胶原蛋白的表达。更多还原

【Abstract】 Pulmonary fibrosis is a chronic lung disease caused by a variety of reasons and characterized in persistent lung injury, repair, remodeling of the organizational structure and extra cellular matrix (mainly collagen typeⅠand typeⅢ) deposition associated with inflammatory response in lung. Recently,study hotspot focuses on the role of cytokines in forming and development of pulmonary fibrosis, including transforming growth factor-β, platelet derived growth factor, connective tissue growth factor, insulin-like growth factor-1 and so on. TGF-βis one of more extensive and indepth study cytokines, including TGF-β1, TGF-β2 and TGF-β3 three forms,which are basically the same biological characteristics. However,TGF-β1 is the most important among them. TGF-β1 can effectively stimulate fibroblast cell division, proliferation and extracellular matrix synthesis and secretion.TGF-βsignal transduction includes two main pathways,Smads protein family and mitogen activated protein kinase (MAPK) pathway. There are four subfamilies in MAPK including signal-regulated kinase (ERK1/2), c-Jun N-terminal kinase (JNK), mitogen-activated protein kinase (P38) and extracellular signal regulated kinase (ERK5). In which,JNK pathway plays a pivotal role in various physiological and pathological events. In recent years, studies have confirmed that JNK signal transduction involved in forming and development of fibrosis in the heart, liver, brain and other organs.N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) is a physiological hemoregulatory inhibitor. Recent study found that AcSDKP could inhibit the interstitial cell proliferation and collagen deposition in kidney and heart, and weakened cardiac and renal fibrosis. In previous experiments,we have confirmed that AcSDKP has an effect on anti-fibrosis because it could inhibit the expression of TGF-β1 and synthesis of collagen in the lung tissue in rat with silicosis. However,we don’t know that AcSDKP whether plays a role in pulmonary fibrosis through regulation of the signal transduction pathways. This study will investigated whether TGF-β1 can induced pulmonary fibroblast proliferation and collagen synthesis through activation of JNK pathways, as well as AcSDKP whether can inhibit pulmonary fibroblast proliferation and collagen synthesis by blocking activation of this pathway.Objectives1. To explore the role in proliferation and collagen synthesis of pulmonary fibroblasts through activation of JNK pathway mediated by TGF-β1.2. To investigate the effect of AcSDKP on inhibiting proliferation and collagen synthesis of pulmonary fibroblast through regulation of JNK pathway mediated by TGF-β1.3. To provide some theory and experimental data in mechanism of AcSDKP anti-fibrosis in lung.Methods1. Pulmonary fibroblasts from newborn Wistar rat were isolated in primary generation and fourth generation of cells was used for experiments.2. The proliferation of pulmonary fibroblasts was detected by MTT assay.3. The localization and distribution of phosphorylation-JNK were observed by confocal laser scanning microscopy.4. The expressions of JNK protein,phosphorylation-JNK protein,c-jun protein,typeⅠand typeⅢcollagen protein in pulmonary fibroblasts were measured with western blot analysis and immunocytochemistry.5. Experimental Groups①0.4% serum group(Control): 0.4% serum in DMEM as a basis medium.②TGF-β1 stimulating group(TGF-β1): TGF-β1 (5μg/L) in basis medium incubated cells for 40 min, to observe the effect of TGF-β1 on stimulating proliferation and collagen synthesis in pulmonary fibroblasts.③SP600125 treating group(SP600125): SP600125 (10nmol/L) in basis medium pre-incubated cells for 45 minutes, then added TGF-β1(5μg/L)in medium and incubated cells for 40 min,to observe the effect of SP600125 on inhibiting proliferation and collagen synthesis and activation of JNK pathway in pulmonary fibroblasts mediated by TGF-β1.④AcSDKP treating group(AcSDKP): AcSDKP (10-8 mol/L) in basis medium pre-incubated cells for 45 minutes,then added TGF-β1(5μg/L)in medium and incubated cells for 40 min,to observe the effect of AcSDKP on inhibiting proliferation and collagen synthesis and activation of JNK pathway in pulmonary fibroblasts mediated by TGF-β1.6. Results were analyzed statistically by single-factor analysis of variance.Results1. Results of MTT assay showed that 1×105/ml density was optimum growth density for pulmonary fibroblasts. The strongest stimulation effect of TGF-β1 in cell proliferation was at 5μg /L in 0.4% serum culture conditions. SP600125(10nmol/L) and AcSDKP(10-8 mol/L) inhibited pulmonary fibroblast proliferation induced by TGF-β1.2. Results of confocal laser scanning microscopy displayed that expression of phosphorylation-JNK labed fluorescence was located more in the cytoplasm in control group. Compared with control group, expression of phosphorylation-JNK decreased in the cytoplasm and increased in nuclei in the TGF-β1 stimulating group. The ratio of nuclei to cytoplasm of phosphorylation-JNK fluorescence density increased in TGF-β1 groups. Compared with the TGF-β1 stimulating group, the fluorescence density in cytoplasm increased and the ratio of nuclei to cytoplasm of fluorescence density decreased in AcSDKP treating group and SP600125 treating group.3. Compared with control group, the expression of phosphorylation-JNK, c-jun and typeⅠand typeⅢcollagen protein increased significantly in TGF-β1 group with western blot analysis. Compared with TGF-β1 group, the expression of phosphorylation-JNK,c-jun and typeⅠand typeⅢcollagen protein decreased in AcSDKP treating group and SP600125 treating group, while the protein levels of JNK were not changed in four groups.ConclusionTGF-β1 could promote pulmonary fibroblast proliferation and typeⅠand typeⅢcollagen expression by activation of JNK pathway. However, AcSDKP could inhibit the proliferation and typeⅠand typeⅢcollagen expression of pulmonary fibroblasts through blocking activation of JNK pathway mediated by TGF-β1.更多还原