Mitofusin2基因抑制大鼠血管平滑肌细胞A7r5增殖的研究

发布时间：2018-05-27 06:23

  本文选题：线粒体融合蛋白基因2 + A7r5细胞　； 参考：《宁夏医科大学》2011年硕士论文

【摘要】：5目的血管平滑肌细胞(vascular smooth muscle cell,VSMC)异常增殖在血管增生性疾病中扮演着十分重要的角色,是动脉粥样硬化形成、高血压、冠心病、经皮冠状动脉腔内成形术后再狭窄的病理基础。细胞生长因子、炎症因子、血流动力学异常等通过相关的细胞信号转导路径,特别是通过激活Ras原癌基因及其所介导的Ras-Raf-MAPK和Ras-PI3K-Akt信号转导路径,引起VSMC过度增殖。因此,阻扰这些路径,抑制细胞异常增殖,是防治血管增生性疾病的有效途径。线粒体融合蛋白2(mitofusin2, Mfn2)基因是近年来发现的一种新型的增殖抑制基因,它不仅抑制多种肿瘤细胞的增殖,并且参与线粒体的融合、调节线粒体的新陈代谢、维持线粒体的网状结构。在高血压大鼠和球囊损伤后再狭窄大鼠主动脉VSMC中Mfn2的表达均下降,因此上调Mfn2的表达可能会抑制该病理过程。本研究拟应用基因克隆和基因重组等方法,观察外源性Mfn2基因对A7r5细胞增殖的影响,并对其分子机制进行探讨,以期为mfn2成为血管增殖性疾病的治疗靶点提供实验依据。 方法通过RT-PCR获得大鼠Mfn2 CDS序列的cDNA,扩增、纯化、回收Mfn2基因片段并与载体pGEM-T连接成重组克隆载体pGEM-T-mfn2,转化入E.coli DH5α感受态细胞大量扩增并行氨苄青霉素筛选。用HindⅢ和BamHⅠ双酶切扩增的大鼠Mfn2基因片段和质粒pEGFP-N1,再将回收的pEGFP-N1大片段与Mfn2基因片段重组,转化感受态DH5α。采用质粒小提产物PCR、HindⅢ和BamHⅠ双酶切和序列分析方法,确定目标片段的存在和序列正确性。将测序正确的重组质粒pEGFP-mfn2在阳离子脂质体的介导下体外转染A7r5细胞,将细胞分成三组:空白对照组A7r5(未转染组),空载体对照组(pEGFP-N1组,转染空质粒),实验组(A7r5-Mfn2-GFP组,转染pEGFP-mfn2)。24h后在荧光显微镜下观察绿色荧光蛋白(green fluorescence protein,GFP)表达情况,分别于转染后48h收获三组细胞,经RT-PCR、Western blot方法检测Mfn2在A7r5细胞的表达情况。转染成功且效率稳定后,通过细胞计数法、MTS法检测Mfn2对A7r5增殖的影响。流式细胞术分析外源性Mfn2基因在体外对A7r5细胞周期分布的影响。Western blot检测三组细胞的磷酸化Raf、磷酸化ERK1/2和磷酸化AKT蛋白表达的变化,采用方差分析对数据进行统计学处理。 结果从A7r5细胞总RNA中经RT-PCR扩增出一条约2.2kb的片段。重组质粒转化感受态DH5α后,通过抗性基因筛选出阳性克隆。质粒小提产物PCR显示存在2.2kb的特异性条带;酶切结果显示重组质粒被切成4.7kb大小的pEGFP-N1载体和2.2kb大小的目的片段;经测序,目的片段的序列与GeneBank中大鼠Mfn2基因的编码序列完全一致;进一步证实成功地构建了含大鼠Mfn2基因的重组真核表达载体。荧光显微镜下观察,转染成功的A7r5细胞中有GFP表达,发出特异性的荧光。按转染率=暗视野所见发绿色荧光的细胞数/明视野所见细胞总数×100%,计算转染率为70%。RT-PCR及Western blot证实转染pEGFP-mfn2组的A7r5细胞中Mfn2mRNA和蛋白表达较对照两组高。细胞计数法、MTS法检测转染pEGFP-mfn2组细胞数明显低于空白对照组和空载体对照组(P0.05),而对照两组细胞数无明显差异。转染后48h流式细胞仪检测结果表明实验组中多数VSMC停滞于GI期,细胞比例为61.733±3.755,与对照组相比差异有统计学意义(F=109.8,P0.05),两对照组相比差异无统计学意义。Western blot结果显示:实验组细胞与两对照组细胞相比,转染后48h磷酸化c-Raf(p-c-Raf),磷酸化ERK1/2(p-ERK1/2)和磷酸化AKT(p-AKT)的表达水平明显降低,其差异有显著性(P0.05)。 结论 1. Mfn2基因过表达可以明显抑制A7r5细胞的增殖。 2. Mfn2基因抑制A7r5细胞增殖的机制可能是通过抑制Ras-Raf-ERK1/2和Ras-PI3K-AKT信号通路,下调磷酸化Raf-1蛋白,磷酸化ERK1/2蛋白和磷酸化AKT蛋白的表达来实现的。
[Abstract]:The abnormal proliferation of vascular smooth muscle cells (vascular smooth muscle cell, VSMC) plays a very important role in vascular proliferative diseases. It is the pathological basis of atherosclerotic formation, hypertension, coronary heart disease, and restenosis after percutaneous coronary angioplasty. Cell growth factors, inflammatory factors, hemodynamic abnormalities, etc. Through the related cell signal transduction pathway, especially by activating the Ras proto oncogene and its mediated Ras-Raf-MAPK and Ras-PI3K-Akt signal transduction pathway, it causes VSMC overproliferation. Therefore, it is an effective way to prevent these pathways and inhibit abnormal proliferation of cells. The mitochondrial fusion protein 2 (mitofusin2, Mfn2) is an effective way to prevent the proliferation of vascular diseases. The gene is a new type of proliferation inhibition gene found in recent years. It not only inhibits the proliferation of many tumor cells, but also participates in mitochondrial fusion, regulates mitochondrial metabolism and maintains the mitochondrial network structure. The expression of Mfn2 in the aorta VSMC of the hypertensive rats and the restenosis rats after the balloon injury is reduced. The expression of Mfn2 may inhibit the pathological process. This study intends to use the methods of gene cloning and gene recombination to observe the effect of exogenous Mfn2 gene on the proliferation of A7r5 cells, and to discuss its molecular mechanism, in order to provide the experimental basis for the target of the treatment of Mfn2 as a vascular proliferative disease.
Methods the cDNA of rat CDS sequence was obtained by RT-PCR, amplification, purification, recovery of Mfn2 gene fragment and pGEM-T-mfn2 of recombinant cloning vector with carrier pGEM-T, and transformed into E.coli DH5 alpha receptive cells to enlarge and parallel ampicillin screening. The rat Mfn2 gene fragments and plasmids were amplified by Hind III and BamH I. Then the reclaimed pEGFP-N1 fragment was reorganized with the Mfn2 gene fragment and transformed into the receptive DH5 alpha. The existence and sequence correctness of the target fragment were determined by the method of double enzyme digestion and sequence analysis of plasmid small extract product PCR, Hind III and BamH I, and transfection of the correct recombinant plasmid pEGFP-mfn2 to A7r5 cells in vitro mediated by cationic liposomes. The cells were divided into three groups: the blank control group A7r5 (untransfected group), the empty body control group (pEGFP-N1 group, transfected empty plasmid), the experimental group (group A7r5-Mfn2-GFP, transfected pEGFP-mfn2).24h after the fluorescence microscope to observe the expression of green fluorescent protein (green fluorescence protein, GFP), and then harvested three groups of cells in 48h after transfection, RT-PCR, West. Ern blot method was used to detect the expression of Mfn2 in A7r5 cells. After the transfection was successful and the efficiency was stable, the effect of Mfn2 on A7r5 proliferation was detected by cell counting and MTS. Flow cytometry analysis of the effect of exogenous Mfn2 gene on the distribution of A7r5 cell cycle in vitro.Western blot detection of phosphorylated Raf, phosphorylated phosphoric acid and phosphoric acid in three groups of cells The variance of AKT protein expression was analyzed by variance analysis.
Results a fragment of the treaty 2.2Kb was amplified by RT-PCR from the total RNA of A7r5 cells. After the recombinant plasmid transformed the receptive DH5 a, the positive clones were screened by the resistance gene. The plasmid small product PCR showed the specific band of 2.2Kb, and the recombinant plasmid was cut into the target fragment of the small pEGFP-N1 carrier and 2.2Kb size of 4.7kb. After sequencing, the sequence of the target fragment was identical with the encoding sequence of the Mfn2 gene in GeneBank, and further confirmed that the recombinant eukaryotic expression vector containing the rat Mfn2 gene was successfully constructed. Under the fluorescence microscope, the transfected A7r5 cells had GFP expression and specific fluorescence. The number of cells in the cell number / visual field was 100%, and the transfection rate of 70%.RT-PCR and Western blot proved that the expression of Mfn2mRNA and protein in the A7r5 cells transfected from pEGFP-mfn2 group was higher than that of the control group. The number of cells in the transfected pEGFP-mfn2 group by MTS method was significantly lower than that of the empty white control group and the empty carrier control group (P0.05), but the number of cells in the transfected pEGFP-mfn2 group was significantly lower than that of the empty carrier control group (P0.05), and the number of cells transfected by MTS method was significantly lower than that of the empty vector control group. There was no significant difference in the number of cells in the two groups. The results of 48h flow cytometry after transfection showed that most of the VSMC in the experimental group stagnated in the GI phase, the proportion of the cells was 61.733 + 3.755, compared with the control group, the difference was statistically significant (F=109.8, P0.05), and there was no statistically significant difference between the two control groups and the difference between the experimental group and the two control group. The expression levels of 48h phosphorylated c-Raf (p-c-Raf), phosphorylated ERK1/2 (p-ERK1/2) and phosphorylated AKT (p-AKT) were significantly lower in the group cells than in the transfected cells, and the difference was significant (P0.05).
conclusion
1. overexpression of Mfn2 can significantly inhibit the proliferation of A7r5 cells.
The mechanism of inhibiting the proliferation of A7r5 cells by 2. Mfn2 gene may be realized by inhibiting the Ras-Raf-ERK1/2 and Ras-PI3K-AKT signaling pathways, down regulating the expression of phosphorylated Raf-1 protein, phosphorylated ERK1/2 protein and phosphorylated AKT protein.
【学位授予单位】：宁夏医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363

【参考文献】

相关期刊论文 前10条

1 李鹏飞,郭艳红,李黔,姚蓬英,陈光慧;腺病毒介导的rHSG-1基因转染对自发性高血压大鼠血管平滑肌细胞增殖的影响[J];北京大学学报(医学版);2004年03期

2 陈春蕾;沈涛;郑铭;郭艳红;朱小君;陈光慧;;线粒体融合蛋白Mfn2对心肌细胞肥大的抑制作用[J];北京大学学报(医学版);2008年05期

3 廖华;曹文静;陈莉莉;陈光慧;郭小梅;;腺病毒介导的线粒体融合素基因-2诱导大鼠颈动脉球囊损伤后血管平滑肌细胞凋亡[J];中华急诊医学杂志;2006年06期

4 李田昌，佟利家，庞永正，刘秀华，王雪清，胡大一，唐朝枢;丝裂素活化蛋白激酶参与内皮素刺激的兔主动脉平滑肌细胞增生[J];生理学报;1996年04期

5 夏耘;吴亚群;何小军;龚建平;裘法祖;;Effects of Mitofusin-2 Gene on Cell Proliferation and Chemotherapy Sensitivity of MCF-7[J];Journal of Huazhong University of Science and Technology(Medical Sciences);2008年02期

6 李俐;王剑明;陈光慧;郭小梅;;Mfn2基因对大鼠血管平滑肌细胞凋亡的促进作用及其机制[J];华中科技大学学报(医学版);2007年01期

7 刘成;陈莉莉;王剑明;曹文静;陈光慧;郭小梅;;Mfn2基因抑制Ras-PI3K-Akt信号途径并诱导大鼠血管平滑肌细胞凋亡[J];华中科技大学学报(医学版);2007年02期

8 陈光慧,刘乃奎,周爱儒,唐朝枢,马大龙,汤健;高血压相关基因(HRG-1)在心血管病发病中的作用(英文)[J];Chinese Medical Journal;2001年08期

9 王贵松,陈光慧,郭艳红,李黔,霍勇,高炜;腺病毒介导高血压相关基因转移对兔颈动脉球囊损伤后新生内膜形成的抑制作用[J];中华心血管病杂志;2004年07期

10 刘亚萍;温绍君;刘雅;赵莉敏;郭艳红;陈新军;王佐广;刘洁琳;文杰;王世奇;汤健;;增殖抑制基因在原发性高血压患者血管平滑肌细胞增殖中的作用[J];中华心血管病杂志;2007年10期

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