【摘要】：目的研究外源性線粒體融合蛋白2基因(Mitofusin2,Mfn2)對大鼠主動脈平滑肌細胞A7r5凋亡的影響，并對其可能的分子機制進行探討。 方法將重組質粒pEGFP-mfn2在陽離子脂質體Lipofectamine2000的介導下體外轉染A7r5細胞,將細胞分為三組：空白對照組（control組，未轉染）、空載體對照組（pEGFP N1組，轉染空質粒）和實驗組（pEGFP mfn2組，轉染pEGFP mfn2）。轉染后，用流式細胞儀（FCM）測定轉染效率。分別于轉染后48h收獲三組細胞，采用Westernblot檢測Mfn2的表達。采用Hoechst染色激光共聚焦顯微鏡、Annexin V-PE/7-AAD雙染流式細胞儀及一步TUNEL細胞凋亡檢測法觀察Mfn2對A7r5凋亡的影響并計算凋亡率。Western Blot方法檢測三組細胞中凋亡相關因子Bcl-xl、Bax、Caspase-9以及磷酸化Akt（p-Akt）和磷酸化Bad(p-Bad)的表達。采用方差分析對數(shù)據(jù)進行統(tǒng)計學處理。 結果以流式細胞儀測定轉染效率，pEGFP N1組和pEGFP mfn2組于轉染48h轉染效率達到最高，分別為（63.3±2.4）%和（65.2±3.5）%，兩組比較差異無顯著性（P＞0.05）。Western blot檢測顯示，pEGFP mfn2組Mfn2蛋白表達水平增高,其半定量結果為（1.084±0.057），control組和pEGFP N1組mfn2蛋白表達半定量分別為（0.534±0.075）、（0.552±0.034），pEGFP mfn2組與兩對照組比較差異均有顯著性（均P0.05）。Hoechst染色激光共聚焦顯微鏡觀察顯示，轉染后48h，pEGFP mfn2組細胞凋亡率為（13.37±2.85）%，與control組（0.92±0.05）%和pEGFP N1組（1.33±0.06）%相比差異均有統(tǒng)計學意義（均P0.05）。Annexin V-PE/7-AAD雙染流式細胞儀分析結果顯示：轉染48h及72h，pEGFP mfn2組細胞凋亡率分別為（15.74±2.42）%、（32.23±3.91）%，空白對照組和空載體對照組細胞均未發(fā)生明顯凋亡，，前者與后二者比較差異均有統(tǒng)計學意義（均P0.01）。轉染72h一步TUNEL法熒光顯微鏡觀察，實驗組可見標記紅色熒光的凋亡細胞，其陽性細胞數(shù)為（21.3±2.8）%，兩對照組未見明顯標記紅色熒光的晚期凋亡細胞。轉染后48h經(jīng)Western blot檢測顯示：Bax蛋白的半定量結果pEGFP mfn2組為（0.879±0.126），與control組（0.545±0.069）和pEGFP N1組（0.498±0.074）比較均有統(tǒng)計學差異（均P0.05）；Bcl-xl蛋白的半定量結果pEGFP mfn2組為（0.527±0.061），與control組（0.992±0.107）和pEGFP N1組（1.011±0.093）比較均有統(tǒng)計學差異（均P0.05）；pEGFP-mfn2組Caspase-9蛋白表達半定量結果為(0.398±0.047)，control組和pEGFP-N1組未見表達；pEGFP mfn2組p-Akt蛋白表達量（0.572±0.085）低于control組（0.943±0.109）和pEGFP N1組（0.935±0.072），其差異有顯著性（均P0.05）；pEGFP mfn2組p-Bad的表達量（0.411±0.046）較control組（0.787±0.125）和pEGFP N1組（0.831±0.114）低，其差異有統(tǒng)計學顯著性（均P0.05）。 結論 1. Mfn2基因過表達可以促進A7r5細胞凋亡。 2. Mfn2基因促進A7r5細胞凋亡可能與抑制Ras-PI3K-Akt信號途徑并活化線粒體凋亡途徑有關，是通過下調P-Akt、P-Bad及Bcl-xl蛋白和上調Bax蛋白從而激活Caspase9來實現(xiàn)的。
[Abstract]:Objective to investigate the effect of exogenous mitochondrial fusion protein 2 (Mitofusin2,Mfn2) gene on A7r5 apoptosis in rat aortic smooth muscle cells and its possible molecular mechanism. Methods the recombinant plasmid pEGFP-mfn2 was transfected into A7r5 cells mediated by cationic liposome Lipofectamine2000 in vitro. The cells were divided into three groups: blank control group (control group, untransfected group), empty vector control group (pEGFP N1group, transfected empty plasmid group) and experimental group (pEGFP mfn2 group, transfected pEGFP mfn2). After transfection, the transfection efficiency was measured by flow cytometry (FCM). Three groups of cells were harvested 48 hours after transfection and the expression of Mfn2 was detected by Westernblot. The effect of Mfn2 on A7r5 apoptosis was observed by Hoechst staining laser confocal microscopy, Annexin V-PE/7-AAD double staining flow cytometry and one step TUNEL cell apoptosis assay. The apoptosis-related factors Bcl-xl,Bax,Caspase-9 and phosphorus in three groups were detected by. Western Blot method. The expression of phosphorylated Akt (p-Akt) and phosphorylated Bad (p-Bad). ANOVA was used to process the data statistically. Results the transfection efficiency was detected by flow cytometry. The transfection efficiency of pEGFP N1-group and pEGFP mfn2 group was the highest (63.3 鹵2.4)% and (65.2 鹵3.5)% respectively at 48 h after transfection. There was no significant difference between the two groups (P > 0. 05). Western blot test showed that the expression of Mfn2 protein in pEGFP mfn2 group was higher than that in pEGFP mfn2 group). The semi-quantitative results were (1.084 鹵0.057), control) and (0.534 鹵0.075), () (0.552 鹵0.034), pEGFP) mfn2 and (0.534 鹵0.075), (0.552 鹵0.034), pEGFP mfn2, respectively. There were significant differences between the two groups (P0.05). The apoptotic rate of pEGFP mfn2 group was (13.37 鹵2.85)% at 48h after transfection, which was significantly higher than that of control group (0.92 鹵0.05)% and pEGFP group N1 (1.33 鹵0.06)% (P0.05). Annexin V-PE/7-AAD double-staining flow cytometry analysis showed that the apoptotic rate was (15.74 鹵2.42)% and (32.23 鹵3.91)% in 48 h and 72 h pEGFP mfn2 group, respectively. There was no obvious apoptosis in the cells of radiation group and empty carrier control group. There was significant difference between the former and the latter (P 0.01). The apoptotic cells labeled with red fluorescence were found in the experimental group (21.3 鹵2.8)%, but no late apoptotic cells marked with red fluorescence were found in the two control groups. 48 hours after transfection, Western blot showed that the semi-quantitative result of Bax protein in pEGFP mfn2 group was (0.879 鹵0.126), which was significantly higher than that in control group (0.545 鹵0.069) and pEGFP N _ 1 group (0.498 鹵0.074) (P0.05). The semi-quantitative results of Bcl-xl protein in pEGFP mfn2 group were (0.527 鹵0.061), which were significantly higher than those in control group (0.992 鹵0.107) and pEGFP N _ 1 group (1.011 鹵0.093) (P0.05), but the semi-quantitative results of Caspase-9 protein expression in pEGFP-mfn2 group were (0.398 鹵0.047), control group and (0.398 鹵0.047), control group. The expression of p-Akt protein in mfn2 group (0.572 鹵0.085) was lower than that in control group (0.943 鹵0.109) and pEGFP group N1 (0.935 鹵0.072), the difference was significant (P 0.05). The expression of p-Bad in); pEGFP mfn2 group (0.411 鹵0.046) was significantly lower than that in control group (0.787 鹵0.125) and pEGFP N1-group (0.831 鹵0.114) (P0.05). Conclusion 1. Overexpression of Mfn2 gene can promote apoptosis of A7r5 cells. 2. Mfn2 gene may promote apoptosis of A7r5 cells by inhibiting Ras-PI3K-Akt signaling pathway and activating mitochondrial apoptotic pathway, which is realized by down-regulating P-AkttPad and Bcl-xl proteins and up-regulating Bax protein to activate Caspase9.
【學位授予單位】：寧夏醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R363

【參考文獻】

相關期刊論文 前10條

1 夏耘;吳亞群;張林;李小蘭;袁惠玲;何小軍;陶德定;龔建平;裘法祖;;線粒體融合素基因-2對人乳腺癌MCF-7細胞株增殖與化療敏感性的影響[J];癌癥;2007年08期

2 郭艷紅;李黔;于海奕;高煒;;增殖抑制基因誘導血管平滑肌細胞凋亡[J];北京大學學報(醫(yī)學版);2007年04期

3 毋麗娜;李志新;盧恬;馬騰;邱曉彥;;人增殖抑制基因(hHSG)聯(lián)合熱處理對結腸癌細胞系HT-29的體外誘導凋亡效應[J];北京大學學報(醫(yī)學版);2008年05期

4 楊世明;孫瑜;涂志業(yè);糜濤;陳莉莉;郭小梅;;EPO對缺血/再灌注損傷大鼠心肌細胞Mfn2蛋白表達及心肌細胞凋亡的影響[J];中國組織化學與細胞化學雜志;2009年06期

5 任澎;李U

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