【作者】 赵丽；

【导师】 郭小梅；

【作者基本信息】 华中科技大学 ， 内科学， 2009， 博士

【摘要】 第一部分重组tMfn2基因腺病毒表达载体的包装及其在VSMCs中的表达1．目的采用脂质体(Lipofectamine^TM 2000)转染方法将携带tMfn2基因的腺病毒骨架质粒包装成重组腺病毒（Adv-tMfn2），并在体外培养大鼠血管平滑肌细胞（VSMC）中表达，观察基因转染情况。2．方法利用Lipofectamine^TM 2000法将经PacI线性化的pAd-tMfn2转染HEK 293A细胞进行包装，待大部分细胞变圆、漂浮后收集细胞。PCR方法证实为复制缺陷型tMfn2基因重组腺病毒，之后进行大量扩增。采用氯化铯梯度离心法纯化病毒，将病毒液进行分装，-80℃保存。利用分光光度计测定重组腺病毒滴度。采用Western blot方法检测tMfn2目的基因在VSMCs中的表达。3．结果融合基因序列正确，PCR可检测到1．3kb的特异性条带并排除野生病毒株。腺病毒Adv-tMfn2滴度为66．424×10⁹pfu/ml。Western blot结果表明Adv-tMfn2感染VSMCs后能有效表达出相应的蛋白。4．结论利用脂质体和293A细胞可在体外方便、快捷地构建携带tMfn2基因的复制缺陷型腺病毒，易进行扩增纯化，从而获得大量高滴度病毒液。tMfn2基因可在VSMCs中高水平表达出目的蛋白，为进一步研究其对VSMCs增殖和凋亡的影响奠定了基础。第二部分tMfn2基因对大鼠血管平滑肌细胞增殖的影响1．目的比较大鼠线粒体融合素基因2（Mfn2）和去除穿膜区序列的线粒体融合素基因2（tMfn2）对大鼠血管平滑肌细胞（VSMCs）增殖的作用，探讨tMfn2基因对VSMCs增殖的影响及其相关的信号通路。2．方法用携带tMfn2基因和Mfn2基因的重组腺病毒（Adv-tMfn2和Adv-Mfn2）感染VSMCs，检测tMfn2蛋白和Mfn2蛋白在细胞中的表达水平；细胞计数法、四甲基偶氮唑盐（MTT）法检测VSMCs的增殖；流式细胞仪检测tMfn2基因对VSMCs细胞周期的影响；Western blot方法分析各组磷酸化ERK1/2和磷酸化Raf-1蛋白表达水平的变化；采用方差分析对数据进行统计学处理。3．结果Adv-tMfn2和Adv-Mfn2感染VSMCs后能有效表达出相应蛋白；细胞计数和MTT结果显示，tMfn2和Mfn2均使VSMCs增殖受到明显抑制（P＜0．01），且前者较后者抑制效果更明显（P＜0．01）；流式细胞仪检测结果表明tMfn2和Mfn2使多数VSMCs停滞于G0/G1期，细胞比例为88．01±4．38％和67．43±6．21％，可见tMfn2基因对细胞周期的影响更明显（P＜0．01）；进一步研究表明tMfn2和Mfn2降低VSMCs磷酸化ERK1/2（p-ERK1/2）和磷酸化Raf-1（p-Raf-1）的表达水平（P＜0．01），且tMfn2作用更明显（P＜0．01）。4．结论与Mfn2基因相比，tMfn2基因更能显著抑制VSMCs增殖，使细胞周期停滞于G0/G1期，这一作用主要是通过抑制Ras-Raf-ERK1/2信号通路，下调磷酸化Raf-1蛋白表达，进而抑制ERK1/2的磷酸化而实现。第三部分tMfn2基因对大鼠血管平滑肌细胞凋亡的影响1．目的线粒体融合素基因2（Mfn2）对大鼠血管平滑肌细胞（VSMCs）的增殖和凋亡都有影响，因此推测去除穿膜区序列的大鼠线粒体融合素基因2（tMfn2）对VSMCs凋亡可能也有作用，本文进一步探讨tMfn2基因对VSMCs凋亡的影响和相关信号通路。2．方法用Adv-tMfn2和Adv-Mfn2分别感染VSMCs，检测tMfn2蛋白和Mfn2蛋白在细胞中的表达水平。采用流式细胞术、细胞凋亡ELISA、TUNEL染色等方法检测tMfn2对VSMCs凋亡的影响；Western blot分析磷酸化Akt（p-Akt）蛋白和线粒体凋亡路径中Bcl-2、Bax、cleaved Caspase-9蛋白表达变化。3．结果Adv-tMfn2和Adv-Mfn2感染VSMCs后能有效表达出相应蛋白；流式细胞仪检测和ELISA结果表明，tMfn2组的凋亡细胞明显较Mfn2组增多，且呈时间依赖性，随着时间的延长，tMfn2促凋亡作用增强（P＜0．01）；TUNEL染色发现tMfn2组的棕黄色颗粒明显多于Mfn2组，说明前者促凋亡作用更显著（P＜0．01）；Western blot结果显示，tMfn2和Mfn2组中p-Akt水平均明显降低，但前者作用更强（P＜0．01）；进一步检测线粒体凋亡路径中的相关蛋白，tMfn2组的Bax蛋白表达显著升高、Bcl-2蛋白表达显著降低，且cleaved caspase-9的活性明显增强，较Mfn2诱导凋亡的作用更强（P＜0．01）。4．结论tMfn2基因同样显著诱导VSMCs凋亡，且其作用随着时间的延长而增强。tMfn2基因的作用主要是通过抑制Ras-PI3K-Akt信号路径来实现，可显著抑制Akt磷酸化，从而激活线粒体凋亡途径，导致VSMCs凋亡。更多还原

【Abstract】 PartⅠPacking,Identification and Expression of the RecombinedAdenovirus containing tMfn2 gene1.ObjectiveTo construct the recombinant adenovirus vector containing the cDNA for Mfn2 with thetransmembrane domain deleted （tMfn2） using the Lipofecamine^TM 2000 Packing System,and examine its ability to express the tMfn2 gene in VSMCs.2.MethodsHEK 293A cells were transfected by using Lipofectamine^TM 2000 and packaged for therecombinant adenovirus particles.Replication-deficient recombinant adenovirus whichcarried the tMfn2 gene were identified by PCR and amplified.The titer of adenovirus wasmeasured by cesium chloride （CsCl） gradient ultracentrifugation.Finally,the expression oftMfn2 gene in VSMCs was determined by Western blot analysis.3.ResultsThe replication-deficient recombinant adenovirus which carried tMfn2 gene wereconstructed successfully and identified by PCR.The titer of adenovirus particles was66.424×10⁹ pfu/ml.The result of Western blot indicated that VSMCs can express highlevel of tMfn2 protein efficiently when transfected.4.ConclusionsThe method of packing in vitro is a shortcut for construction of replication-deficientrecombinant adenovirus with tMfn2 gene,which provides a basis for the further research onthe effect of tMfn2 gene on the proliferation and apoptosis of VSMCs. PartⅡEffects of tMfn2 gene on inhibiting the proliferation of VSMCs1.ObjectiveTo investigate the effect of tMfn2 on inhibiting the proliferation of VSMCs.2.MethodsVSMCs were infected by adenovirus-mediated tMfn2 （Adv-tMfn2） or adenovirus-mediated Mfn2 （Adv-Mfn2）.Western blot was used to testify the expression of these twoproteins.Cell counting and MTT were used to investigate the effect on proliferation ofVSMCs and Flow cytometry to observe the cell cycle.Before collected,the cells werestimulated by ET-1 for 2～5min.Western blot were used to analyze the expression ofp-ERK1/2 and p-Raf-l.ANOVA was used to make data analysis.3.ResultsAccording to the results of cell counting and MTT,they both indicated that Adv-tMfn2had more remarkable effect on inhibiting the proliferation of VSMCs than Adv-Mfn2 （P＜0.01）.Flow cytometry showed that most of the cells infected by Adv-tMfn2 or Adv-Mfn2were blocked in the stage of G0/G1 and few cells entered into the S phase;tMfn2 hadstronger effect than Mfn2 （P＜0.01）.The result of Western blot indicated that tMfn2 caninhibit phosphorylation of ERK1/2 and Raf-1 protein more effectively compared with Mfn2（P＜0.01）.4.ConclusionsOverexpression of tMfn2 reduces ET-1-stimulated VSMCs proliferation by inhibitingERK phosphorylation and Raf activity,then leading to the arrest of cell cycle.What’s moreimportant,tMfn2 gene is superior to Mfn2 gene in attenuating the proliferation of VSMCsvia the Ras-ERK1/2-Raf signaling pathway. PartⅢtMfn2 gene promotes apoptosis of VSMCs via mitochondrialpathway1.ObjectiveThe aim of the study is to investigate the effect of tMfn2 gene on promoting theapoptosis of VSMCs.2.MethodsVSMCs were infected by adenovirus-mediated tMfn2 （Adv-tMfn2） or adenovirus-mediated Mfn2 （Adv-Mfn2）.FACS analysis,cell death ELISA and TUNEL staining wereused to investigate the role of tMfn2 on VSMCs apoptosis.Western blot was used toanalyze the expression of p-Akt,Bcl-2,Bax and cleaved capase-9.3.ResultsFACS analyses and ELISA assay showed tMfn2 was superior to Mfn2 in promoting theapoptosis of VSMCs and had a robust time-dependent increase in apoptosis （P＜0.01）.TUNEL staining displayed that there were more positive-apoptotic VSMCs in tMfn2 groupthan that in Mfn2 group （P＜0.01）.The results of Western blot indicated that the proteinexpression of phosphorylated Akt and Bcl-2 remarkably decreased,whereas Bax andcleaved capased-9 protein highly expressed in tMfn2-infected group.4.ConclusionsThe proapoptotic effect of tMfn2 was mediated by inhibition of PI3K-Akt signallingand subsequently activated the mitochondrial apoptotic pathway.To compare with Mfn2gene,tMfn2 has more remarkable effect on inducing VSMCs apoptosis.更多还原