發(fā)布時間：2018-01-13 21:02

  本文關鍵詞：心肌纖維化中Ⅰ型纖維狀膠原蛋白經(jīng)由α2β1整合素介導的成纖維細胞表型轉(zhuǎn)化相關分子通路機制研究　出處：《南京醫(yī)科大學》2017年博士論文　論文類型：學位論文

  更多相關文章： 纖維狀膠原蛋白 α2β1整合素 PTEN PP2A 心肌纖維化

【摘要】：背景成纖維細胞分化成特異性表達α-SMA的肌成纖維細胞并分泌大量細胞外基質(zhì)是心肌纖維化的標志。而纖維狀Ⅰ型膠原蛋白是心肌纖維化中主要的細胞外基質(zhì)。纖維狀Ⅰ型膠原蛋白和成纖維細胞在心肌纖維化發(fā)生發(fā)展中相互作用,互為因果,進一步加重心肌纖維化進程。在其中α2β1整合素作為細胞受體介導成纖維細胞和細胞外基質(zhì)間的"交流"。然而目前為止,纖維狀Ⅰ型膠原蛋白(FC)如何經(jīng)由α2β1整合素調(diào)控成纖維細胞的機制仍無定論。為此我們在體外建立三維細胞模型,模擬心梗后體內(nèi)纖維化環(huán)境,從而觀察纖維化Ⅰ型膠原蛋白對成纖維細胞表型及α2β1整合素相關分子信號通路影響。目的探討纖維狀Ⅰ型膠原經(jīng)由α2β1整合素介導成纖維細胞表型轉(zhuǎn)化的相關分子通路機制材料方法1.成纖維細胞分離與培養(yǎng)心臟成纖維細胞從3月齡C57BL/6雄性小鼠心臟分離提取。本試驗采用第三代細胞。2.三維纖維狀Ⅰ型膠原蛋白培養(yǎng)基建立將Ⅰ型膠原蛋白溶液(1.25ml)中加入0.5ml的5×DMEM,并通過1%胎牛血清將其稀釋至1×DMEM。3.細胞系GD25是null-α2β1整合素的成纖維細胞,GD25α2β1 integrin細胞是GD25細胞重組α2β1整合素在成纖維細胞中的表達。4.細胞遷移試驗。細胞遷移試驗是以細胞阻抗傳感(ECIS)(Applied BioPhysics,Troy,NY,USA)為基礎實時研究組織培養(yǎng)中細胞活性的方法。遷移由獨立評估測量持續(xù)20小時。5.脈沖追蹤法PTEN基因以35S蛋氨酸定量標記并以beta-actin進行免疫印跡測定蛋白的表達。6.PP2A磷酸酶活性測定PP2A的活性根據(jù)制造商的指示(Millipore,Temecula,CA,USA)通過磷酸化K-RpT-I-R-R進行去磷酸化評估。7.增殖試驗細胞接種于含10%胎牛血清的培養(yǎng)基中培養(yǎng)1天。第4天,細胞被胰蛋白酶消化和計數(shù)后收獲。8.數(shù)據(jù)分析所有實驗最低重復三次。數(shù)據(jù)表示為平均值。免疫印跡的數(shù)據(jù)由BioSpectrum Imaging 系統(tǒng)(UVP,Upland,CA,USA)測定。所有的結(jié)果進行雙側(cè)分析,未配對t檢驗。P0.05具有統(tǒng)計學意義。結(jié)果1.纖維狀Ⅰ型膠原蛋白促進成纖維細胞分化及增殖為了探究纖維狀Ⅰ型膠原蛋白對成纖維細胞的影響,將成纖維細胞種植在FC中。形態(tài)學觀察示FC中的成纖維細胞形態(tài)較對照組更加纖長,通過細胞增殖能力及移形能力檢測,發(fā)現(xiàn)FC中的成纖維細胞明顯高于普通對照組,α-SMA的表達明顯增高。同時將FC,PP2A抑制劑,TGFβ1這些因素同時處理細胞,結(jié)果提示相比其他因素FC促進成纖維細胞分化及增殖能力更強。2.纖維狀Ⅰ型膠原蛋白降低成纖維細胞中a2β1整合素及PTEN表達western blot提示FC中的成纖維細胞a2β1整合素、PTEN含量明顯少于普通對照組,而p-Akt及α-SMA則有所增高。分離心梗模型小鼠中梗死區(qū)域及非梗死區(qū)域成纖維細胞,結(jié)果提示梗死區(qū)域肌成纖維細胞中β1整合素明顯低于正常組織成纖維細胞。接著通過半定量RT-PCR方法分別檢測了 FC及TC中成纖維細胞a2β1整合素及PTENmRNA的含量。結(jié)果示與對照組相比無論a2或β1整合素mRNA含量都明顯下降。然而PTEN mRNA的含量在兩組間卻沒有明顯改變。進一步我們通過Pulse-Chase assay方法檢測PTEN蛋白穩(wěn)定性,結(jié)果提示我們與正常對照組相比FC中的PTEN含量明顯降低。3.敲低a2β1整合素可以促進纖維狀Ⅰ型膠原介導肌成纖維細胞分化及增殖低表達β1整合素的成纖維細胞在FC中的增殖能力明顯高于正常對照組,其PTEN含量明顯降低,而其下游通路p-Akt含量及α-SMA含量明顯增高。為了進一步明確PTEN的功能,通過RNA干擾技術敲低和過表達成纖維細胞中PTEN的含量。對于敲低組,與正常對照組相比在FC中的增殖能力明顯增高(p0.001),而a2β1整合素蛋白與對照組相比表達沒有明顯改變,但p-Akt及α-SMA蛋白表達增高。4.纖維狀Ⅰ型膠原介導a2β1整合素調(diào)節(jié)PP2A活性而PP2A是否參與到Ⅰ型膠原經(jīng)由a2β1整合素介導的成纖維細胞表型轉(zhuǎn)化中?。通過對PP2A活性檢測發(fā)現(xiàn)與對照組相比FC中的成纖維細胞中PP2A蛋白活性明顯降低(p0.002),接著我們敲低成纖維細胞中β1整合素表達。結(jié)果顯示低表達β1整合素PP2A活性明顯低于正常對照組(p0.004)我們分別將GD25 α2β1 integrin-null(GD25)成纖維細胞和 GD25 α2β1 integrin 重組成纖維細胞種植在FC中,GD25成纖維細胞內(nèi)PP2A的活性顯著降低。而GD25 α2β1 integrin重組成纖維細胞中PP2A的活性明顯增高(5-fold)。5.PP2A調(diào)節(jié)纖維狀Ⅰ型膠原介導的成纖維細胞表型轉(zhuǎn)化通過對細胞增殖分析,發(fā)現(xiàn)無論OA+組還是shRNA PP2Ac組成纖維細胞在FC中增殖明顯高于對照組。shRNAPP2Ac組細胞中PP2Ac表達較對照組相比下降79%,然而PTEN表達卻輕微減少;同時我們有趣的發(fā)現(xiàn)shRNAPP2Ac組細胞中p-Akt含量卻明顯升高(3.8-fold),同時也觀測到α-SMA含量明顯增加。接著我們將成纖維細胞種植在FC上,用OA處理細胞,同時設立對照組,觀測0小時,0.5小時變化。結(jié)果提示0.5小時后OA(+)及OA(-)成纖維細胞中PTEN較0小時相比表達減少。然而0.5小時OA(+)及OA(-)成纖維細胞兩組間PTEN表達差異不明顯,同時OA(+)組中p-Akt和α-SMA表達卻顯著升高(分別3.93fold,5.7fold)。上述結(jié)果提示我PP2A主要靶點是Akt而不是PTEN。我們將種植在FC上的成纖維細胞分為三組,分別為予wortmannin(an AKT inhibitor),U0126(anERKinhibitor),wortmannin+U0126處理,抑制ERK后,細胞增殖輕微減少(p0.05),而抑制了 AKT后,細胞增殖顯著減少(p0.01),而如果同時抑制ERK和AKT后細胞增殖減少更加明顯(p0.001)。以上結(jié)果提示我們相比于單獨ERK通路,Akt通路在成纖維細胞分化和增殖調(diào)控中起的作用更明顯。結(jié)論本研究證明了纖維狀Ⅰ型膠原蛋白可以經(jīng)由低表達的α2β1 integrin促進成纖維細胞向肌成纖維細胞表型轉(zhuǎn)化。而AKT的過分激活,可能與PTEN及PP2A低活性相關。因此,我們可以認為α2β1integrin/PTEN/PP2A信號通路在纖維狀Ⅰ型膠原介導的成纖維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.

【學位授予單位】：南京醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R54

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