TRPM7对人胚肺成纤维细胞增殖及分化的调控机制研究

发布时间：2018-05-21 19:37

本文选题：瞬时受体电位通道7 + 肺纤维化　；参考：《安徽医科大学》2013年硕士论文

【摘要】：肺纤维化(pulmonary fibrosis，PF)是一种严重的肺间质疾病，主要病理特点是早期的弥漫性肺泡炎，后期大量成纤维细胞病理性增殖转型及细胞外基质(extracellular matrix，ECM)进行性异常积聚并取代正常的肺组织结构~[1]。肺纤维化发病受多种因素的共同影响，尤其是大量细胞因子的刺激作用，使成纤维细胞（FB）不断地增殖和转型为肌成纤维细胞(myofibroblast，MB)~[2]。流行病学调查发现，肺纤维化的发病率、死亡率不断上升，而且随着年龄的增长，其发病率和死亡率也逐渐升高。目前，对PF的药物治疗主要以糖皮质激素和免疫抑制剂为主，但效果均不理想[3]。深入探讨其发病机制，寻找新的药物靶点，一直是国内外药物研究的热点。瞬时受体电位通道（transient receptor potential channels, TRP channel）是位于细胞膜上的一类重要的阳离子通道，瞬时受体电位通道7(TRPM7)是TRP家族成员之一，是一种具有阳离子通道和蛋白激酶双重结构的双功能膜蛋白，可使自身或底物磷酸化，也被称为“通道酶”~[4,5]。文献报道，TRPM7广泛存在于机体组织中，参与细胞的生长、增殖、迁移、凋亡等多种生理病理过程~[6,7]，并在纤维化疾病的发病过程中具有重要作用~[8,9]。但其在肺纤维化中的作用还未见文献报道。本实验选用人胚肺成纤维细胞系MRC-5为研究对象，观察TRPM7对MRC-5细胞增殖及分化的影响，并探讨其可能的作用机制。主要研究内容概括如下： 1. TRPM7在人胚肺成纤维细胞系MRC-5中的表达本实验通过TGF-β1刺激人胚肺成纤维细胞MRC-5，RT-PCR和Western blot检测MRC-5中TRPM7的表达情况。结果显示，TGF-β1刺激组，TRPM7的表达明显高于正常组。 2.抑制TRPM7表达对人胚肺成纤维细胞系MRC-5增殖及分化的影响采用MTT比色法测定TRPM7非特异性阻断剂Gd~(3+)或2-APB对MRC-5的增殖抑制率；流式细胞术检测MRC-5周期变化；RT-PCR检测TRPM7、α-SMA、Collagen mRNA的表达变化；Western blot检测α-SMA、Collagen蛋白的表达水平。结果显示，Gd~(3+)或2-APB能够不同程度的降低MRC-5中TRPM7mRNA的表达，并且在一定范围内抑制MRC-5的增殖。进一步研究TRPM7对MRC-5细胞周期的影响，结果发现，TRPM7阻断剂Gd~(3+)或2-APB可通过促使细胞聚积于G_0/G_1期，降低G_2/M期及S期细胞，影响MRC-5细胞的增殖。同时，Gd~(3+)或2-APB能够降低MRC-5中α-SMA、Collagen的mRNA和蛋白的表达，表明下调TRPM7的表达可抑制MRC-5的增殖和分化。利用Lipofectamine~(TM)2000将特异性的TRPM7-siRNA转染到MRC-5中，下调TRPM7在人胚肺成纤维细胞系MRC-5中的表达。分别运用流式细胞术检测MRC-5细胞周期的影响；RT-PCR及Western blot法检测TRPM7、α-SMA、Collagen mRNA和蛋白的表达。结果提示，与对照组相比，，TRPM7-siRNA转染组细胞增殖受到抑制，肺纤维化标志性蛋白α-SMA、Collagen的表达显著下降。 3. TRPM7对人胚肺成纤维细胞系MRC-5中PI3K/AKT信号通路的影响给予TGF-β1体外诱导肺纤维化模型，再分别给予PI3K/AKT通路特异性阻断剂LY294002、离子通道阻断剂Gd~(3+)、2-APB处理，MTT检测细胞增殖抑制率，Western blot法检测p-Akt、t-Akt蛋白的表达。结果提示，与正常组比较，TGF-β1明显促进MRC-5的增殖，而给予LY294002干预后，MRC-5的增殖明显受到抑制，同时TGF-β1能显著促进MRC-5p-Akt蛋白的表达，当使用LY294002、Gd~(3+)、2-APB处理后，p-Akt蛋白表达均受到抑制，t-Akt蛋白表达无明显变化，更进一步研究发现，TRPM7-siRNA转染组p-Akt蛋白表达均受到抑制，t-Akt表达无明显变化。以上结果表明：下调TRPM7表达可以抑制MRC-5的增殖及分化，对肺纤维化具有一定的保护作用，其机制可能与调控PI3K/AKT信号通路有关。
[Abstract]:Pulmonary fibrosis (PF) is a serious pulmonary interstitial disease. The main pathological features are early diffuse alveolitis, a large number of fibroblasts in the later stage and the abnormal accumulation of extracellular matrix (extracellular matrix, ECM) in a large number of fibroblasts and the generation of normal lung tissue structure ~[1]. pulmonary fibrosis. The common effects of the elements, especially the stimulation of a large number of cytokines, make the fibroblasts (FB) proliferate and transform into myofibroblast (myofibroblast, MB) ~[2]. epidemiological survey, and find that the incidence and mortality of pulmonary fibrosis are increasing, and the incidence and mortality of the pulmonary fibrosis are increasing with age. The main drug treatment for PF is glucocorticoid and immunosuppressant, but the effect is not ideal [3]. to explore its pathogenesis and find new drug targets. It has always been a hot spot of drug research at home and abroad.
Transient receptor potential channels (TRP channel) is an important class of cationic channels on the cell membrane. Instantaneous receptor potential channel 7 (TRPM7) is one of the members of the TRP family. It is a bifunctional membrane protein with the dual structure of cationic and protein kinase, which can phosphorylate itself or substrate. Also known as the "channel enzyme" ~[4,5]. literature, TRPM7 widely exists in the body tissues and participates in many physiological and pathological processes, such as cell growth, proliferation, migration, apoptosis and other physiological and pathological processes, ~[6,7], and plays an important role in the pathogenesis of fibrotic disease, but its role in pulmonary fibrosis has not been reported in literature.
In this experiment, the human embryo lung fibroblast cell line MRC-5 was selected as the research object to observe the effect of TRPM7 on the proliferation and differentiation of MRC-5 cells and to explore the possible mechanism of action. The main contents are as follows:
Expression of 1. TRPM7 in human embryonic lung fibroblast cell line MRC-5
In this experiment, the expression of TRPM7 in MRC-5, RT-PCR and Western blot was detected by TGF- beta 1 stimulation of human embryonic lung fibroblasts (MRC-5, RT-PCR and Western). The results showed that the expression of TRPM7 was significantly higher in the TGF- beta 1 stimulation group than in the normal group.
2. inhibition of TRPM7 expression on proliferation and differentiation of human embryonic lung fibroblast cell line MRC-5
MTT colorimetric assay was used to determine the proliferation inhibition rate of TRPM7 non specific blocking agent Gd~ (3+) or 2-APB; flow cytometry was used to detect MRC-5 cycle changes; RT-PCR was used to detect the expression of TRPM7, alpha -SMA, Collagen mRNA. The expression of TRPM7mRNA in low MRC-5 and the inhibition of the proliferation of MRC-5 in a certain range. Further study the effect of TRPM7 on the cycle of MRC-5 cells. It is found that the TRPM7 blocker Gd~ (3+) or 2-APB can induce cells to accumulate in the G_0/G_1 period, reduce the G_2/M phase and S phase cells, and affect the proliferation of the cells. The expression of -SMA and Collagen mRNA and protein in C-5 showed that down regulation of TRPM7 expression could inhibit MRC-5 proliferation and differentiation.
The specific TRPM7-siRNA was transfected into MRC-5 by Lipofectamine~ (TM) 2000, and the expression of TRPM7 in the human embryo lung fibroblast cell line MRC-5 was downregulated. The effects of flow cytometry on the detection of MRC-5 cell cycle were used respectively. RT-PCR and Western blot methods were used to detect TRPM7, alpha -SMA, expression and protein expression. The results suggested that the results were compared with the control group. In TRPM7-siRNA transfection group, cell proliferation was inhibited, and the expression of -SMA and Collagen in lung fibrosis decreased significantly.
Effect of 3. TRPM7 on PI3K/AKT signaling pathway in human embryonic lung fibroblast cell line MRC-5
The pulmonary fibrosis model was induced by TGF- beta 1 in vitro, then PI3K/AKT pathway specific blocker LY294002, ion channel blocker Gd~ (3+), 2-APB treatment, MTT detection of cell proliferation inhibition rate, Western blot method to detect p-Akt, t-Akt protein expression. 4002 the proliferation of MRC-5 was obviously inhibited, and TGF- beta 1 could significantly promote the expression of MRC-5p-Akt protein. When LY294002, Gd~ (3+), 2-APB were used, the expression of p-Akt protein was inhibited, and the expression of t-Akt protein was not obviously changed. Further research found that the expression of p-Akt protein in the TRPM7-siRNA transfected group was inhibited and t-Akt expression was expressed. There was no obvious change.
The above results suggest that down regulation of TRPM7 can inhibit the proliferation and differentiation of MRC-5, and has a protective effect on pulmonary fibrosis, and its mechanism may be related to the regulation of PI3K/AKT signaling pathway.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R563.9;R96

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