本文選題：線粒體融合蛋白基因2 + A7r5細胞　； 參考：《寧夏醫(yī)科大學》2011年碩士論文

【摘要】：5目的血管平滑肌細胞(vascular smooth muscle cell,VSMC)異常增殖在血管增生性疾病中扮演著十分重要的角色,是動脈粥樣硬化形成、高血壓、冠心病、經皮冠狀動脈腔內成形術后再狹窄的病理基礎。細胞生長因子、炎癥因子、血流動力學異常等通過相關的細胞信號轉導路徑,特別是通過激活Ras原癌基因及其所介導的Ras-Raf-MAPK和Ras-PI3K-Akt信號轉導路徑,引起VSMC過度增殖。因此,阻擾這些路徑,抑制細胞異常增殖,是防治血管增生性疾病的有效途徑。線粒體融合蛋白2(mitofusin2, Mfn2)基因是近年來發(fā)現(xiàn)的一種新型的增殖抑制基因,它不僅抑制多種腫瘤細胞的增殖,并且參與線粒體的融合、調節(jié)線粒體的新陳代謝、維持線粒體的網狀結構。在高血壓大鼠和球囊損傷后再狹窄大鼠主動脈VSMC中Mfn2的表達均下降,因此上調Mfn2的表達可能會抑制該病理過程。本研究擬應用基因克隆和基因重組等方法,觀察外源性Mfn2基因對A7r5細胞增殖的影響,并對其分子機制進行探討,以期為mfn2成為血管增殖性疾病的治療靶點提供實驗依據(jù)。 方法通過RT-PCR獲得大鼠Mfn2 CDS序列的cDNA,擴增、純化、回收Mfn2基因片段并與載體pGEM-T連接成重組克隆載體pGEM-T-mfn2,轉化入E.coli DH5α感受態(tài)細胞大量擴增并行氨芐青霉素篩選。用HindⅢ和BamHⅠ雙酶切擴增的大鼠Mfn2基因片段和質粒pEGFP-N1,再將回收的pEGFP-N1大片段與Mfn2基因片段重組,轉化感受態(tài)DH5α。采用質粒小提產物PCR、HindⅢ和BamHⅠ雙酶切和序列分析方法,確定目標片段的存在和序列正確性。將測序正確的重組質粒pEGFP-mfn2在陽離子脂質體的介導下體外轉染A7r5細胞,將細胞分成三組:空白對照組A7r5(未轉染組),空載體對照組(pEGFP-N1組,轉染空質粒),實驗組(A7r5-Mfn2-GFP組,轉染pEGFP-mfn2)。24h后在熒光顯微鏡下觀察綠色熒光蛋白(green fluorescence protein,GFP)表達情況,分別于轉染后48h收獲三組細胞,經RT-PCR、Western blot方法檢測Mfn2在A7r5細胞的表達情況。轉染成功且效率穩(wěn)定后,通過細胞計數(shù)法、MTS法檢測Mfn2對A7r5增殖的影響。流式細胞術分析外源性Mfn2基因在體外對A7r5細胞周期分布的影響。Western blot檢測三組細胞的磷酸化Raf、磷酸化ERK1/2和磷酸化AKT蛋白表達的變化,采用方差分析對數(shù)據(jù)進行統(tǒng)計學處理。 結果從A7r5細胞總RNA中經RT-PCR擴增出一條約2.2kb的片段。重組質粒轉化感受態(tài)DH5α后,通過抗性基因篩選出陽性克隆。質粒小提產物PCR顯示存在2.2kb的特異性條帶;酶切結果顯示重組質粒被切成4.7kb大小的pEGFP-N1載體和2.2kb大小的目的片段;經測序,目的片段的序列與GeneBank中大鼠Mfn2基因的編碼序列完全一致;進一步證實成功地構建了含大鼠Mfn2基因的重組真核表達載體。熒光顯微鏡下觀察,轉染成功的A7r5細胞中有GFP表達,發(fā)出特異性的熒光。按轉染率=暗視野所見發(fā)綠色熒光的細胞數(shù)/明視野所見細胞總數(shù)×100%,計算轉染率為70%。RT-PCR及Western blot證實轉染pEGFP-mfn2組的A7r5細胞中Mfn2mRNA和蛋白表達較對照兩組高。細胞計數(shù)法、MTS法檢測轉染pEGFP-mfn2組細胞數(shù)明顯低于空白對照組和空載體對照組(P0.05),而對照兩組細胞數(shù)無明顯差異。轉染后48h流式細胞儀檢測結果表明實驗組中多數(shù)VSMC停滯于GI期,細胞比例為61.733±3.755,與對照組相比差異有統(tǒng)計學意義(F=109.8,P0.05),兩對照組相比差異無統(tǒng)計學意義。Western blot結果顯示:實驗組細胞與兩對照組細胞相比,轉染后48h磷酸化c-Raf(p-c-Raf),磷酸化ERK1/2(p-ERK1/2)和磷酸化AKT(p-AKT)的表達水平明顯降低,其差異有顯著性(P0.05)。 結論 1. Mfn2基因過表達可以明顯抑制A7r5細胞的增殖。 2. Mfn2基因抑制A7r5細胞增殖的機制可能是通過抑制Ras-Raf-ERK1/2和Ras-PI3K-AKT信號通路,下調磷酸化Raf-1蛋白,磷酸化ERK1/2蛋白和磷酸化AKT蛋白的表達來實現(xiàn)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.
【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】：2011
【分類號】：R363

【參考文獻】

相關期刊論文 前10條

1 李鵬飛,郭艷紅,李黔,姚蓬英,陳光慧;腺病毒介導的rHSG-1基因轉染對自發(fā)性高血壓大鼠血管平滑肌細胞增殖的影響[J];北京大學學報(醫(yī)學版);2004年03期

2 陳春蕾;沈濤;鄭銘;郭艷紅;朱小君;陳光慧;;線粒體融合蛋白Mfn2對心肌細胞肥大的抑制作用[J];北京大學學報(醫(yī)學版);2008年05期

3 廖華;曹文靜;陳莉莉;陳光慧;郭小梅;;腺病毒介導的線粒體融合素基因-2誘導大鼠頸動脈球囊損傷后血管平滑肌細胞凋亡[J];中華急診醫(yī)學雜志;2006年06期

4 李田昌，佟利家，龐永正，劉秀華，王雪清，胡大一，唐朝樞;絲裂素活化蛋白激酶參與內皮素刺激的兔主動脈平滑肌細胞增生[J];生理學報;1996年04期

5 夏耘;吳亞群;何小軍;龔建平;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;王劍明;陳光慧;郭小梅;;Mfn2基因對大鼠血管平滑肌細胞凋亡的促進作用及其機制[J];華中科技大學學報(醫(yī)學版);2007年01期

7 劉成;陳莉莉;王劍明;曹文靜;陳光慧;郭小梅;;Mfn2基因抑制Ras-PI3K-Akt信號途徑并誘導大鼠血管平滑肌細胞凋亡[J];華中科技大學學報(醫(yī)學版);2007年02期

8 陳光慧,劉乃奎,周愛儒,唐朝樞,馬大龍,湯健;高血壓相關基因(HRG-1)在心血管病發(fā)病中的作用(英文)[J];Chinese Medical Journal;2001年08期

9 王貴松,陳光慧,郭艷紅,李黔,霍勇,高煒;腺病毒介導高血壓相關基因轉移對兔頸動脈球囊損傷后新生內膜形成的抑制作用[J];中華心血管病雜志;2004年07期

10 劉亞萍;溫紹君;劉雅;趙莉敏;郭艷紅;陳新軍;王佐廣;劉潔琳;文杰;王世奇;湯健;;增殖抑制基因在原發(fā)性高血壓患者血管平滑肌細胞增殖中的作用[J];中華心血管病雜志;2007年10期

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