Mitofusin2基因对大鼠血管平滑肌细胞A7r5凋亡作用的研究
发布时间:2018-10-15 18:44
【摘要】:目的研究外源性线粒体融合蛋白2基因(Mitofusin2,Mfn2)对大鼠主动脉平滑肌细胞A7r5凋亡的影响,并对其可能的分子机制进行探讨。 方法将重组质粒pEGFP-mfn2在阳离子脂质体Lipofectamine2000的介导下体外转染A7r5细胞,将细胞分为三组:空白对照组(control组,未转染)、空载体对照组(pEGFP N1组,转染空质粒)和实验组(pEGFP mfn2组,转染pEGFP mfn2)。转染后,用流式细胞仪(FCM)测定转染效率。分别于转染后48h收获三组细胞,采用Westernblot检测Mfn2的表达。采用Hoechst染色激光共聚焦显微镜、Annexin V-PE/7-AAD双染流式细胞仪及一步TUNEL细胞凋亡检测法观察Mfn2对A7r5凋亡的影响并计算凋亡率。Western Blot方法检测三组细胞中凋亡相关因子Bcl-xl、Bax、Caspase-9以及磷酸化Akt(p-Akt)和磷酸化Bad(p-Bad)的表达。采用方差分析对数据进行统计学处理。 结果以流式细胞仪测定转染效率,pEGFP N1组和pEGFP mfn2组于转染48h转染效率达到最高,分别为(63.3±2.4)%和(65.2±3.5)%,两组比较差异无显著性(P>0.05)。Western blot检测显示,pEGFP mfn2组Mfn2蛋白表达水平增高,其半定量结果为(1.084±0.057),control组和pEGFP N1组mfn2蛋白表达半定量分别为(0.534±0.075)、(0.552±0.034),pEGFP mfn2组与两对照组比较差异均有显著性(均P0.05)。Hoechst染色激光共聚焦显微镜观察显示,转染后48h,pEGFP mfn2组细胞凋亡率为(13.37±2.85)%,与control组(0.92±0.05)%和pEGFP N1组(1.33±0.06)%相比差异均有统计学意义(均P0.05)。Annexin V-PE/7-AAD双染流式细胞仪分析结果显示:转染48h及72h,pEGFP mfn2组细胞凋亡率分别为(15.74±2.42)%、(32.23±3.91)%,空白对照组和空载体对照组细胞均未发生明显凋亡,,前者与后二者比较差异均有统计学意义(均P0.01)。转染72h一步TUNEL法荧光显微镜观察,实验组可见标记红色荧光的凋亡细胞,其阳性细胞数为(21.3±2.8)%,两对照组未见明显标记红色荧光的晚期凋亡细胞。转染后48h经Western blot检测显示:Bax蛋白的半定量结果pEGFP mfn2组为(0.879±0.126),与control组(0.545±0.069)和pEGFP N1组(0.498±0.074)比较均有统计学差异(均P0.05);Bcl-xl蛋白的半定量结果pEGFP mfn2组为(0.527±0.061),与control组(0.992±0.107)和pEGFP N1组(1.011±0.093)比较均有统计学差异(均P0.05);pEGFP-mfn2组Caspase-9蛋白表达半定量结果为(0.398±0.047),control组和pEGFP-N1组未见表达;pEGFP mfn2组p-Akt蛋白表达量(0.572±0.085)低于control组(0.943±0.109)和pEGFP N1组(0.935±0.072),其差异有显著性(均P0.05);pEGFP mfn2组p-Bad的表达量(0.411±0.046)较control组(0.787±0.125)和pEGFP N1组(0.831±0.114)低,其差异有统计学显著性(均P0.05)。 结论 1. Mfn2基因过表达可以促进A7r5细胞凋亡。 2. Mfn2基因促进A7r5细胞凋亡可能与抑制Ras-PI3K-Akt信号途径并活化线粒体凋亡途径有关,是通过下调P-Akt、P-Bad及Bcl-xl蛋白和上调Bax蛋白从而激活Caspase9来实现的。
[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 卤2.4)% and (65.2 卤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 卤0.057), control) and (0.534 卤0.075), () (0.552 卤0.034), pEGFP) mfn2 and (0.534 卤0.075), (0.552 卤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 卤2.85)% at 48h after transfection, which was significantly higher than that of control group (0.92 卤0.05)% and pEGFP group N1 (1.33 卤0.06)% (P0.05). Annexin V-PE/7-AAD double-staining flow cytometry analysis showed that the apoptotic rate was (15.74 卤2.42)% and (32.23 卤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 卤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 卤0.126), which was significantly higher than that in control group (0.545 卤0.069) and pEGFP N _ 1 group (0.498 卤0.074) (P0.05). The semi-quantitative results of Bcl-xl protein in pEGFP mfn2 group were (0.527 卤0.061), which were significantly higher than those in control group (0.992 卤0.107) and pEGFP N _ 1 group (1.011 卤0.093) (P0.05), but the semi-quantitative results of Caspase-9 protein expression in pEGFP-mfn2 group were (0.398 卤0.047), control group and (0.398 卤0.047), control group. The expression of p-Akt protein in mfn2 group (0.572 卤0.085) was lower than that in control group (0.943 卤0.109) and pEGFP group N1 (0.935 卤0.072), the difference was significant (P 0.05). The expression of p-Bad in); pEGFP mfn2 group (0.411 卤0.046) was significantly lower than that in control group (0.787 卤0.125) and pEGFP N1-group (0.831 卤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.
【学位授予单位】:宁夏医科大学
【学位级别】:硕士
【学位授予年份】:2012
【分类号】:R363
[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 卤2.4)% and (65.2 卤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 卤0.057), control) and (0.534 卤0.075), () (0.552 卤0.034), pEGFP) mfn2 and (0.534 卤0.075), (0.552 卤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 卤2.85)% at 48h after transfection, which was significantly higher than that of control group (0.92 卤0.05)% and pEGFP group N1 (1.33 卤0.06)% (P0.05). Annexin V-PE/7-AAD double-staining flow cytometry analysis showed that the apoptotic rate was (15.74 卤2.42)% and (32.23 卤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 卤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 卤0.126), which was significantly higher than that in control group (0.545 卤0.069) and pEGFP N _ 1 group (0.498 卤0.074) (P0.05). The semi-quantitative results of Bcl-xl protein in pEGFP mfn2 group were (0.527 卤0.061), which were significantly higher than those in control group (0.992 卤0.107) and pEGFP N _ 1 group (1.011 卤0.093) (P0.05), but the semi-quantitative results of Caspase-9 protein expression in pEGFP-mfn2 group were (0.398 卤0.047), control group and (0.398 卤0.047), control group. The expression of p-Akt protein in mfn2 group (0.572 卤0.085) was lower than that in control group (0.943 卤0.109) and pEGFP group N1 (0.935 卤0.072), the difference was significant (P 0.05). The expression of p-Bad in); pEGFP mfn2 group (0.411 卤0.046) was significantly lower than that in control group (0.787 卤0.125) and pEGFP N1-group (0.831 卤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.
【学位授予单位】:宁夏医科大学
【学位级别】:硕士
【学位授予年份】:2012
【分类号】:R363
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