發(fā)布時(shí)間：2018-05-27 06:23

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

【摘要】：5目的血管平滑肌細(xì)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(dòng)脈粥樣硬化形成、高血壓、冠心病、經(jīng)皮冠狀動(dòng)脈腔內(nèi)成形術(shù)后再狹窄的病理基礎(chǔ)。細(xì)胞生長(zhǎng)因子、炎癥因子、血流動(dòng)力學(xué)異常等通過(guò)相關(guān)的細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)路徑,特別是通過(guò)激活Ras原癌基因及其所介導(dǎo)的Ras-Raf-MAPK和Ras-PI3K-Akt信號(hào)轉(zhuǎn)導(dǎo)路徑,引起VSMC過(guò)度增殖。因此,阻擾這些路徑,抑制細(xì)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(lái)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(xì)胞的增殖,并且參與線粒體的融合、調(diào)節(jié)線粒體的新陳代謝、維持線粒體的網(wǎng)狀結(jié)構(gòu)。在高血壓大鼠和球囊損傷后再狹窄大鼠主動(dòng)脈VSMC中Mfn2的表達(dá)均下降,因此上調(diào)Mfn2的表達(dá)可能會(huì)抑制該病理過(guò)程。本研究擬應(yīng)用基因克隆和基因重組等方法,觀察外源性Mfn2基因?qū)7r5細(xì)胞增殖的影響,并對(duì)其分子機(jī)制進(jìn)行探討,以期為mfn2成為血管增殖性疾病的治療靶點(diǎn)提供實(shí)驗(yàn)依據(jù)。 方法通過(guò)RT-PCR獲得大鼠Mfn2 CDS序列的cDNA,擴(kuò)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(zhuǎn)化入E.coli DH5α感受態(tài)細(xì)胞大量擴(kuò)增并行氨芐青霉素篩選。用HindⅢ和BamHⅠ雙酶切擴(kuò)增的大鼠Mfn2基因片段和質(zhì)粒pEGFP-N1,再將回收的pEGFP-N1大片段與Mfn2基因片段重組,轉(zhuǎn)化感受態(tài)DH5α。采用質(zhì)粒小提產(chǎn)物PCR、HindⅢ和BamHⅠ雙酶切和序列分析方法,確定目標(biāo)片段的存在和序列正確性。將測(cè)序正確的重組質(zhì)粒pEGFP-mfn2在陽(yáng)離子脂質(zhì)體的介導(dǎo)下體外轉(zhuǎn)染A7r5細(xì)胞,將細(xì)胞分成三組:空白對(duì)照組A7r5(未轉(zhuǎn)染組),空載體對(duì)照組(pEGFP-N1組,轉(zhuǎn)染空質(zhì)粒),實(shí)驗(yàn)組(A7r5-Mfn2-GFP組,轉(zhuǎn)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(dá)情況,分別于轉(zhuǎn)染后48h收獲三組細(xì)胞,經(jīng)RT-PCR、Western blot方法檢測(cè)Mfn2在A7r5細(xì)胞的表達(dá)情況。轉(zhuǎn)染成功且效率穩(wěn)定后,通過(guò)細(xì)胞計(jì)數(shù)法、MTS法檢測(cè)Mfn2對(duì)A7r5增殖的影響。流式細(xì)胞術(shù)分析外源性Mfn2基因在體外對(duì)A7r5細(xì)胞周期分布的影響。Western blot檢測(cè)三組細(xì)胞的磷酸化Raf、磷酸化ERK1/2和磷酸化AKT蛋白表達(dá)的變化,采用方差分析對(duì)數(shù)據(jù)進(jìn)行統(tǒng)計(jì)學(xué)處理。 結(jié)果從A7r5細(xì)胞總RNA中經(jīng)RT-PCR擴(kuò)增出一條約2.2kb的片段。重組質(zhì)粒轉(zhuǎn)化感受態(tài)DH5α后,通過(guò)抗性基因篩選出陽(yáng)性克隆。質(zhì)粒小提產(chǎn)物PCR顯示存在2.2kb的特異性條帶;酶切結(jié)果顯示重組質(zhì)粒被切成4.7kb大小的pEGFP-N1載體和2.2kb大小的目的片段;經(jīng)測(cè)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(jìn)一步證實(shí)成功地構(gòu)建了含大鼠Mfn2基因的重組真核表達(dá)載體。熒光顯微鏡下觀察,轉(zhuǎn)染成功的A7r5細(xì)胞中有GFP表達(dá),發(fā)出特異性的熒光。按轉(zhuǎn)染率=暗視野所見(jiàn)發(fā)綠色熒光的細(xì)胞數(shù)/明視野所見(jiàn)細(xì)胞總數(shù)×100%,計(jì)算轉(zhuǎn)染率為70%。RT-PCR及Western blot證實(shí)轉(zhuǎn)染pEGFP-mfn2組的A7r5細(xì)胞中Mfn2mRNA和蛋白表達(dá)較對(duì)照兩組高。細(xì)胞計(jì)數(shù)法、MTS法檢測(cè)轉(zhuǎn)染pEGFP-mfn2組細(xì)胞數(shù)明顯低于空白對(duì)照組和空載體對(duì)照組(P0.05),而對(duì)照兩組細(xì)胞數(shù)無(wú)明顯差異。轉(zhuǎn)染后48h流式細(xì)胞儀檢測(cè)結(jié)果表明實(shí)驗(yàn)組中多數(shù)VSMC停滯于GI期,細(xì)胞比例為61.733±3.755,與對(duì)照組相比差異有統(tǒng)計(jì)學(xué)意義(F=109.8,P0.05),兩對(duì)照組相比差異無(wú)統(tǒng)計(jì)學(xué)意義。Western blot結(jié)果顯示:實(shí)驗(yàn)組細(xì)胞與兩對(duì)照組細(xì)胞相比,轉(zhuǎn)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(dá)水平明顯降低,其差異有顯著性(P0.05)。 結(jié)論 1. Mfn2基因過(guò)表達(dá)可以明顯抑制A7r5細(xì)胞的增殖。 2. Mfn2基因抑制A7r5細(xì)胞增殖的機(jī)制可能是通過(guò)抑制Ras-Raf-ERK1/2和Ras-PI3K-AKT信號(hào)通路,下調(diào)磷酸化Raf-1蛋白,磷酸化ERK1/2蛋白和磷酸化AKT蛋白的表達(dá)來(lái)實(shí)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(xué)位授予單位】：寧夏醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R363

【參考文獻(xiàn)】

相關(guān)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(dǎo)的rHSG-1基因轉(zhuǎn)染對(duì)自發(fā)性高血壓大鼠血管平滑肌細(xì)胞增殖的影響[J];北京大學(xué)學(xué)報(bào)(醫(yī)學(xué)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(duì)心肌細(xì)胞肥大的抑制作用[J];北京大學(xué)學(xué)報(bào)(醫(yī)學(xué)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(dǎo)的線粒體融合素基因-2誘導(dǎo)大鼠頸動(dòng)脈球囊損傷后血管平滑肌細(xì)胞凋亡[J];中華急診醫(yī)學(xué)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(nèi)皮素刺激的兔主動(dòng)脈平滑肌細(xì)胞增生[J];生理學(xué)報(bào);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?qū)Υ笫笱芷交〖?xì)胞凋亡的促進(jìn)作用及其機(jī)制[J];華中科技大學(xué)學(xué)報(bào)(醫(yī)學(xué)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(hào)途徑并誘導(dǎo)大鼠血管平滑肌細(xì)胞凋亡[J];華中科技大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2007年02期

8 陳光慧,劉乃奎,周愛(ài)儒,唐朝樞,馬大龍,湯健;高血壓相關(guān)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(dǎo)高血壓相關(guān)基因轉(zhuǎn)移對(duì)兔頸動(dòng)脈球囊損傷后新生內(nèi)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(xì)胞增殖中的作用[J];中華心血管病雜志;2007年10期

，

本文編號(hào)：1940821