發(fā)布時間：2018-05-21 19:37

  本文選題：瞬時受體電位通道7 + 肺纖維化��； 參考：《安徽醫(yī)科大學(xué)》2013年碩士論文

【摘要】：肺纖維化(pulmonary fibrosis，PF)是一種嚴重的肺間質(zhì)疾病，主要病理特點是早期的彌漫性肺泡炎，后期大量成纖維細胞病理性增殖轉(zhuǎn)型及細胞外基質(zhì)(extracellular matrix，ECM)進行性異常積聚并取代正常的肺組織結(jié)構(gòu)~[1]。肺纖維化發(fā)病受多種因素的共同影響，尤其是大量細胞因子的刺激作用，使成纖維細胞（FB）不斷地增殖和轉(zhuǎn)型為肌成纖維細胞(myofibroblast，MB)~[2]。流行病學(xué)調(diào)查發(fā)現(xiàn)，肺纖維化的發(fā)病率、死亡率不斷上升，而且隨著年齡的增長，其發(fā)病率和死亡率也逐漸升高。目前，對PF的藥物治療主要以糖皮質(zhì)激素和免疫抑制劑為主，但效果均不理想[3]。深入探討其發(fā)病機制，尋找新的藥物靶點，一直是國內(nèi)外藥物研究的熱點。 瞬時受體電位通道（transient receptor potential channels, TRP channel）是位于細胞膜上的一類重要的陽離子通道，瞬時受體電位通道7(TRPM7)是TRP家族成員之一，是一種具有陽離子通道和蛋白激酶雙重結(jié)構(gòu)的雙功能膜蛋白，可使自身或底物磷酸化，也被稱為“通道酶”~[4,5]。文獻報道，TRPM7廣泛存在于機體組織中，參與細胞的生長、增殖、遷移、凋亡等多種生理病理過程~[6,7]，并在纖維化疾病的發(fā)病過程中具有重要作用~[8,9]。但其在肺纖維化中的作用還未見文獻報道。 本實驗選用人胚肺成纖維細胞系MRC-5為研究對象，觀察TRPM7對MRC-5細胞增殖及分化的影響，并探討其可能的作用機制。主要研究內(nèi)容概括如下： 1. TRPM7在人胚肺成纖維細胞系MRC-5中的表達 本實驗通過TGF-β1刺激人胚肺成纖維細胞MRC-5，RT-PCR和Western blot檢測MRC-5中TRPM7的表達情況。結(jié)果顯示，TGF-β1刺激組，TRPM7的表達明顯高于正常組。 2.抑制TRPM7表達對人胚肺成纖維細胞系MRC-5增殖及分化的影響 采用MTT比色法測定TRPM7非特異性阻斷劑Gd~(3+)或2-APB對MRC-5的增殖抑制率；流式細胞術(shù)檢測MRC-5周期變化；RT-PCR檢測TRPM7、α-SMA、Collagen mRNA的表達變化；Western blot檢測α-SMA、Collagen蛋白的表達水平。結(jié)果顯示，Gd~(3+)或2-APB能夠不同程度的降低MRC-5中TRPM7mRNA的表達，并且在一定范圍內(nèi)抑制MRC-5的增殖。進一步研究TRPM7對MRC-5細胞周期的影響，結(jié)果發(fā)現(xiàn)，TRPM7阻斷劑Gd~(3+)或2-APB可通過促使細胞聚積于G_0/G_1期，降低G_2/M期及S期細胞，影響MRC-5細胞的增殖。同時，Gd~(3+)或2-APB能夠降低MRC-5中α-SMA、Collagen的mRNA和蛋白的表達，表明下調(diào)TRPM7的表達可抑制MRC-5的增殖和分化。 利用Lipofectamine~(TM)2000將特異性的TRPM7-siRNA轉(zhuǎn)染到MRC-5中，下調(diào)TRPM7在人胚肺成纖維細胞系MRC-5中的表達。分別運用流式細胞術(shù)檢測MRC-5細胞周期的影響；RT-PCR及Western blot法檢測TRPM7、α-SMA、Collagen mRNA和蛋白的表達。結(jié)果提示，與對照組相比，，TRPM7-siRNA轉(zhuǎn)染組細胞增殖受到抑制，肺纖維化標(biāo)志性蛋白α-SMA、Collagen的表達顯著下降。 3. TRPM7對人胚肺成纖維細胞系MRC-5中PI3K/AKT信號通路的影響 給予TGF-β1體外誘導(dǎo)肺纖維化模型，再分別給予PI3K/AKT通路特異性阻斷劑LY294002、離子通道阻斷劑Gd~(3+)、2-APB處理，MTT檢測細胞增殖抑制率，Western blot法檢測p-Akt、t-Akt蛋白的表達。結(jié)果提示，與正常組比較，TGF-β1明顯促進MRC-5的增殖，而給予LY294002干預(yù)后，MRC-5的增殖明顯受到抑制，同時TGF-β1能顯著促進MRC-5p-Akt蛋白的表達，當(dāng)使用LY294002、Gd~(3+)、2-APB處理后，p-Akt蛋白表達均受到抑制，t-Akt蛋白表達無明顯變化，更進一步研究發(fā)現(xiàn)，TRPM7-siRNA轉(zhuǎn)染組p-Akt蛋白表達均受到抑制，t-Akt表達無明顯變化。 以上結(jié)果表明：下調(diào)TRPM7表達可以抑制MRC-5的增殖及分化，對肺纖維化具有一定的保護作用，其機制可能與調(diào)控PI3K/AKT信號通路有關(guān)。
[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.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R563.9;R96

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