心肌纤维化中Ⅰ型纤维状胶原蛋白经由α2β1整合素介导的成纤维细胞表型转化相关分子通路机制研究

发布时间：2018-01-13 21:02

本文关键词：心肌纤维化中Ⅰ型纤维状胶原蛋白经由α2β1整合素介导的成纤维细胞表型转化相关分子通路机制研究　出处：《南京医科大学》2017年博士论文　论文类型：学位论文

【摘要】：背景成纤维细胞分化成特异性表达α-SMA的肌成纤维细胞并分泌大量细胞外基质是心肌纤维化的标志。而纤维状Ⅰ型胶原蛋白是心肌纤维化中主要的细胞外基质。纤维状Ⅰ型胶原蛋白和成纤维细胞在心肌纤维化发生发展中相互作用,互为因果,进一步加重心肌纤维化进程。在其中α2β1整合素作为细胞受体介导成纤维细胞和细胞外基质间的"交流"。然而目前为止,纤维状Ⅰ型胶原蛋白(FC)如何经由α2β1整合素调控成纤维细胞的机制仍无定论。为此我们在体外建立三维细胞模型,模拟心梗后体内纤维化环境,从而观察纤维化Ⅰ型胶原蛋白对成纤维细胞表型及α2β1整合素相关分子信号通路影响。目的探讨纤维状Ⅰ型胶原经由α2β1整合素介导成纤维细胞表型转化的相关分子通路机制材料方法1.成纤维细胞分离与培养心脏成纤维细胞从3月龄C57BL/6雄性小鼠心脏分离提取。本试验采用第三代细胞。2.三维纤维状Ⅰ型胶原蛋白培养基建立将Ⅰ型胶原蛋白溶液(1.25ml)中加入0.5ml的5×DMEM,并通过1%胎牛血清将其稀释至1×DMEM。3.细胞系GD25是null-α2β1整合素的成纤维细胞,GD25α2β1 integrin细胞是GD25细胞重组α2β1整合素在成纤维细胞中的表达。4.细胞迁移试验。细胞迁移试验是以细胞阻抗传感(ECIS)(Applied BioPhysics,Troy,NY,USA)为基础实时研究组织培养中细胞活性的方法。迁移由独立评估测量持续20小时。5.脉冲追踪法PTEN基因以35S蛋氨酸定量标记并以beta-actin进行免疫印迹测定蛋白的表达。6.PP2A磷酸酶活性测定PP2A的活性根据制造商的指示(Millipore,Temecula,CA,USA)通过磷酸化K-RpT-I-R-R进行去磷酸化评估。7.增殖试验细胞接种于含10%胎牛血清的培养基中培养1天。第4天,细胞被胰蛋白酶消化和计数后收获。8.数据分析所有实验最低重复三次。数据表示为平均值。免疫印迹的数据由BioSpectrum Imaging 系统(UVP,Upland,CA,USA)测定。所有的结果进行双侧分析,未配对t检验。P0.05具有统计学意义。结果1.纤维状Ⅰ型胶原蛋白促进成纤维细胞分化及增殖为了探究纤维状Ⅰ型胶原蛋白对成纤维细胞的影响,将成纤维细胞种植在FC中。形态学观察示FC中的成纤维细胞形态较对照组更加纤长,通过细胞增殖能力及移形能力检测,发现FC中的成纤维细胞明显高于普通对照组,α-SMA的表达明显增高。同时将FC,PP2A抑制剂,TGFβ1这些因素同时处理细胞,结果提示相比其他因素FC促进成纤维细胞分化及增殖能力更强。2.纤维状Ⅰ型胶原蛋白降低成纤维细胞中a2β1整合素及PTEN表达western blot提示FC中的成纤维细胞a2β1整合素、PTEN含量明显少于普通对照组,而p-Akt及α-SMA则有所增高。分离心梗模型小鼠中梗死区域及非梗死区域成纤维细胞,结果提示梗死区域肌成纤维细胞中β1整合素明显低于正常组织成纤维细胞。接着通过半定量RT-PCR方法分别检测了 FC及TC中成纤维细胞a2β1整合素及PTENmRNA的含量。结果示与对照组相比无论a2或β1整合素mRNA含量都明显下降。然而PTEN mRNA的含量在两组间却没有明显改变。进一步我们通过Pulse-Chase assay方法检测PTEN蛋白稳定性,结果提示我们与正常对照组相比FC中的PTEN含量明显降低。3.敲低a2β1整合素可以促进纤维状Ⅰ型胶原介导肌成纤维细胞分化及增殖低表达β1整合素的成纤维细胞在FC中的增殖能力明显高于正常对照组,其PTEN含量明显降低,而其下游通路p-Akt含量及α-SMA含量明显增高。为了进一步明确PTEN的功能,通过RNA干扰技术敲低和过表达成纤维细胞中PTEN的含量。对于敲低组,与正常对照组相比在FC中的增殖能力明显增高(p0.001),而a2β1整合素蛋白与对照组相比表达没有明显改变,但p-Akt及α-SMA蛋白表达增高。4.纤维状Ⅰ型胶原介导a2β1整合素调节PP2A活性而PP2A是否参与到Ⅰ型胶原经由a2β1整合素介导的成纤维细胞表型转化中?。通过对PP2A活性检测发现与对照组相比FC中的成纤维细胞中PP2A蛋白活性明显降低(p0.002),接着我们敲低成纤维细胞中β1整合素表达。结果显示低表达β1整合素PP2A活性明显低于正常对照组(p0.004)我们分别将GD25 α2β1 integrin-null(GD25)成纤维细胞和 GD25 α2β1 integrin 重组成纤维细胞种植在FC中,GD25成纤维细胞内PP2A的活性显著降低。而GD25 α2β1 integrin重组成纤维细胞中PP2A的活性明显增高(5-fold)。5.PP2A调节纤维状Ⅰ型胶原介导的成纤维细胞表型转化通过对细胞增殖分析,发现无论OA+组还是shRNA PP2Ac组成纤维细胞在FC中增殖明显高于对照组。shRNAPP2Ac组细胞中PP2Ac表达较对照组相比下降79%,然而PTEN表达却轻微减少;同时我们有趣的发现shRNAPP2Ac组细胞中p-Akt含量却明显升高(3.8-fold),同时也观测到α-SMA含量明显增加。接着我们将成纤维细胞种植在FC上,用OA处理细胞,同时设立对照组,观测0小时,0.5小时变化。结果提示0.5小时后OA(+)及OA(-)成纤维细胞中PTEN较0小时相比表达减少。然而0.5小时OA(+)及OA(-)成纤维细胞两组间PTEN表达差异不明显,同时OA(+)组中p-Akt和α-SMA表达却显著升高(分别3.93fold,5.7fold)。上述结果提示我PP2A主要靶点是Akt而不是PTEN。我们将种植在FC上的成纤维细胞分为三组,分别为予wortmannin(an AKT inhibitor),U0126(anERKinhibitor),wortmannin+U0126处理,抑制ERK后,细胞增殖轻微减少(p0.05),而抑制了 AKT后,细胞增殖显著减少(p0.01),而如果同时抑制ERK和AKT后细胞增殖减少更加明显(p0.001)。以上结果提示我们相比于单独ERK通路,Akt通路在成纤维细胞分化和增殖调控中起的作用更明显。结论本研究证明了纤维状Ⅰ型胶原蛋白可以经由低表达的α2β1 integrin促进成纤维细胞向肌成纤维细胞表型转化。而AKT的过分激活,可能与PTEN及PP2A低活性相关。因此,我们可以认为α2β1integrin/PTEN/PP2A信号通路在纤维状Ⅰ型胶原介导的成纤维分化中起到重要的作用。
[Abstract]:The background of fibroblast differentiation specific expression of alpha -SMA fibroblasts and extracellular matrix is a sign of myocardial fibrosis. The fibrous collagen is the main extracellular matrix of myocardial fibrosis. Fibrous collagen and fibroblasts in reciprocal causation of myocardial fibrosis in the development of further interaction. Aggravate the process of myocardial fibrosis. The alpha 2 beta 1 integrin as a cell receptor mediated fibroblast and extracellular matrix between the "exchange". However, so far, fibrous collagen (FC) in the alpha 2 beta 1 integrin regulation mechanism of fiber cells remains uncertain. So we establish a three-dimensional cell model in vitro and in vivo environment simulation of fibrosis after myocardial infarction, to observe fibrosis collagen on fibroblast phenotype and alpha 2 beta 1 integrin molecules related to signal through Objective to investigate the effect of road. Fibrous collagen via alpha 2 beta 1 integrin mediated transformation method into material related molecular fibroblast phenotype pathway 1. fibroblasts isolated and cultured cardiac fibroblasts isolated from March old male C57BL/6 mice heart. The cells of the third generation of.2. three-dimensional fibrillar collagen the establishment of the culture medium of type I collagen solution (1.25ml) by adding 0.5ml 5 * DMEM, and by 1% fetal bovine serum will be diluted to 1 * DMEM.3. GD25 cell line is fibroblast null- alpha 2 beta 1 integrin alpha 2 beta 1, GD25 integrin cells are GD25 cells of recombinant alpha 2 beta 1 integrin in.4. cell migration test expression in fiber cells. Cell migration test is based on the cell impedance sensing (ECIS) (Applied, BioPhysics, Troy, NY, USA) for cell activity in culture based real-time research organization. Migration by independent review Estimation of measurement for 20 hours.5. pulse tracing method of PTEN gene with 35S markers and PP2A.6.PP2A quantitative determination of methionine phosphatase activity according to the manufacturer's instructions to the beta-actin protein expression was measured by Western blot (Millipore, Temecula, CA, USA) through phosphorylation of K-RpT-I-R-R culture medium for 1 days to assess.7. phosphorylation of cell proliferation test inoculation with 10% fetal bovine serum. The cells were fourth days, all the experiments were repeated three times the minimum harvest.8. data analysis of trypsin digestion and counting. Data were expressed as mean. Western blot data by BioSpectrum Imaging system (UVP, Upland, CA, USA) were measured. All the results were analyzed no, paired t test.P0.05 has statistical significance. Results 1. fibrous collagen promote fibroblast proliferation and differentiation in order to explore the fibrous collagen protein of fibroblast Cells, fibroblasts were seeded in FC. Observed in FC fibroblasts compared with the control group by longer, cell proliferation ability and transitional ability testing, found in FC fibroblasts was significantly higher than that of normal control group, the expression of alpha -SMA was significantly increased. At the same time, FC, PP2A inhibitors of TGF beta 1, these factors at the same time cells, results suggest that compared to the other factors FC promote fibroblast differentiation and proliferation ability of.2. fibrous collagen decreased in fibroblasts of A2 integrin beta 1 expression of PTEN and Western blot suggested that fibroblast A2 beta 1 integrin into FC, the content of PTEN was significantly less than the normal control group p-Akt and alpha -SMA increased. The separation in a mouse model of myocardial infarction infarction area and non infarct area fibroblasts, results suggest that the infarct area of myofibroblasts integrin beta 1 was significantly lower in Yu Zheng The normal tissue fibroblasts. Then through semi quantitative RT-PCR method were used to detect the content of fibroblast A2 beta 1 integrin and PTENmRNA FC and TC. Results showed that compared with the control group, both A2 and integrin beta 1 mRNA content decreased significantly. However, the content of PTEN mRNA in the two groups did not significantly change further. We use Pulse-Chase assay method to detect PTEN protein stability, our results suggest that compared with normal control group the content of PTEN FC was significantly decreased in.3. knockdown of A2 beta 1 integrin can promote fibrous collagen mediated myogenic differentiation and proliferation of low expression of integrin beta 1 fibroblast proliferation was obviously in FC higher than the normal control group, the content of PTEN was decreased, and the downstream pathway of p-Akt and the content of alpha -SMA content was significantly increased. In order to further clarify the function of PTEN by RNA interference knockdown and over the table A PTEN content in fiber cells. The knockdown group, compared with normal control group in FC proliferation significantly increased (p0.001), A2 beta 1 integrin protein expression compared with the control group did not change significantly, but the expression of p-Akt and alpha -SMA protein increased.4. fibrous collagen mediated A2 beta 1 integrins regulate the activity of PP2A and PP2A are involved in the type I collagen by A2 beta 1 integrin mediated fibroblast phenotype?. the activity of PP2A was found in FC compared with the control group significantly decreased PP2A protein activity in fibroblasts (p0.002), then we knockdown expression in fibroblasts integrin beta 1. Results show that the low expression of beta 1 integrin PP2A activity was significantly lower than the normal control group (p0.004) we will GD25 alpha 2 beta 1 integrin-null (GD25) fibroblasts and GD25 alpha 2 beta 1 integrin fibroblasts grown in FC, GD25 Fiber intracellular PP2A activity decreased significantly. While the GD25 alpha 2 beta 1 integrin PP2A fiber cell activity increased significantly (5-fold).5.PP2A regulating fibrous collagen mediated fibroblast phenotype by analysis of cell proliferation, found both in the OA+ group and shRNA PP2Ac in FC fibroblasts proliferation the control group was significantly higher than that in.ShRNAPP2Ac cells compared to the control group, the expression of PP2Ac decreased by 79%, however, the expression of PTEN was slightly reduced; at the same time we find interesting content of p-Akt cells in shRNAPP2Ac group were significantly increased (3.8-fold), also observed alpha -SMA content increased significantly. Then we fibroblasts were seeded on FC, treatment cells with OA, and the control group, the observation of 0 hours, 0.5 hours and 0.5 hours after the change. The results showed that OA (+) and OA (-) in fibroblasts of PTEN is 0 hours compared to 0.5 hours. However, the reduced expression of O A (+) and OA (-) fibroblasts between the two groups was no significant difference in the expression of PTEN, OA (+) was significantly increased in p-Akt and -SMA expression group (3.93fold, 5.7fold). These results suggest that I PP2A main target is Akt instead of PTEN. we will be planted in the FC fiber cells were divided into three groups, respectively, with wortmannin (an AKT inhibitor), U0126 (anERKinhibitor), wortmannin+U0126 treatment, the inhibition of ERK cell proliferation after a slight decrease (P0.05), and AKT was inhibited, cell proliferation was significantly reduced (P0.01), and at the same time if inhibition of ERK cell proliferation after AKT and decreased more obviously (p0.001). These results suggest that we compared to the single ERK pathway, Akt pathway in the differentiation and proliferation of fibroblasts in the regulation of the role of the more obvious. Conclusion this study demonstrated that the fibrous collagen through the low expression of alpha 2 beta 1 integrin promote fibroblast to muscle The excessive activation of AKT may be related to the low activity of PTEN and PP2A. Therefore, we can think that the alpha 2 beta 1integrin/PTEN/PP2A signaling pathway plays an important role in fibroid type collagen mediated fibroblast differentiation.

【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R54

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