tMfn2基因对大鼠血管平滑肌细胞增殖的作用与机制研究
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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66．424×10~9pfu/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。
PartⅠ
Packing,Identification and Expression of the RecombinedAdenovirus containing tMfn2 gene
1.Objective
To 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.Methods
HEK 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.Results
The replication-deficient recombinant adenovirus which carried tMfn2 gene wereconstructed successfully and identified by PCR.The titer of adenovirus particles was66.424×10~9 pfu/ml.The result of Western blot indicated that VSMCs can express highlevel of tMfn2 protein efficiently when transfected.
4.Conclusions
The 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.
PartⅡ
Effects of tMfn2 gene on inhibiting the proliferation of VSMCs
1.Objective
To investigate the effect of tMfn2 on inhibiting the proliferation of VSMCs.
2.Methods
VSMCs 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.Results
According 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.Conclusions
Overexpression 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.
PartⅢ
tMfn2 gene promotes apoptosis of VSMCs via mitochondrialpathway
1.Objective
The aim of the study is to investigate the effect of tMfn2 gene on promoting theapoptosis of VSMCs.
2.Methods
VSMCs 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.Results
FACS 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.Conclusions
The 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.
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66．424×10~9pfu/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。
PartⅠ
Packing,Identification and Expression of the RecombinedAdenovirus containing tMfn2 gene
1.Objective
To 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.Methods
HEK 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.Results
The replication-deficient recombinant adenovirus which carried tMfn2 gene wereconstructed successfully and identified by PCR.The titer of adenovirus particles was66.424×10~9 pfu/ml.The result of Western blot indicated that VSMCs can express highlevel of tMfn2 protein efficiently when transfected.
4.Conclusions
The 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.
PartⅡ
Effects of tMfn2 gene on inhibiting the proliferation of VSMCs
1.Objective
To investigate the effect of tMfn2 on inhibiting the proliferation of VSMCs.
2.Methods
VSMCs 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.Results
According 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.Conclusions
Overexpression 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.
PartⅢ
tMfn2 gene promotes apoptosis of VSMCs via mitochondrialpathway
1.Objective
The aim of the study is to investigate the effect of tMfn2 gene on promoting theapoptosis of VSMCs.
2.Methods
VSMCs 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.Results
FACS 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.Conclusions
The 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.
引文
1. Chen KH, Guo X, Ma D, et al. Dysregulation of HSG triggers vascular proliferation disorder. Nat Cell Biol 2004,6(9):872-883.
2. Chien KR and Hoshijima M. Unravelling Ras signals in cardiovascular disease. Nat Cell Biol 2004,6:807-808.
3. Guo XM, Chen KH, Guo YH, et al. Mitofusin2 triggers vascular smooth muscle cell apoptosis via mitochondrial death pathway. Circ Res 2007,101 (11):1113-1122.
4. Majhen D, Ambriovic-Ristov A. Adenoviral vectors-How to use them in cancer gene therapy? Virus Res 2006, 119(2): 121-133.
5. Fang L, Moore XL, Gao XM, et al. Down-regulation of mitofusin-2 expression in cardiac hypertrophy in vitro and in vivo. Life Sciences 2007, 80: 2154 -2160.
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7. Douglas JT. Adenoviral vectors for gene threapy. Mol Biotechnol 2007,36(1):71 -80.
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15．Widmann C．Gibson S，Jarpe MB，et al．Mitogen-activated protein kinase：onservation of a three-kinase module from yeast to human．Pysiol Rev 1999，79(1)：143-180．
16．Garrington TP，Johnson GL．Organization and regulation of mitogen-activated protein kinase signaling pathway．Curr Opin Cell 1999，11(2)：211-218．
17．Melina PK．Timthy RB，Tzeng E．Gene therapy for restenosis．Circ Res 2000， 86：829-833．
18．Guo YH， Chen KH， GAO W， et al．Overexpression of Mitofusin 2 inhibited oxidized low-density lipoprotein induced vascular smooth muscle cell proliferation and reduced atherosclerotic lesion formation in rabbit．Biochem Biophys Res Commun 2007，363(2)：411-417
19．廖华，曹文静，陈莉莉，等．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中华急诊医学杂志2006，15(6)：506-509．
20．Datta SR， Dudek H， Tao X， et al．Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery．Cell 1997， 91(2)： 231-241．
21．Cantley LC．The phosphoinositide 3-Kinase pathway．Science 2002， 296(5573)： 1655-1657．
22．Lange H， Suryapranata H，De Luca G， et al．Folate therapy and in-stent restenosis after coronary stenting．N Engl J Med 2004， 350(26)： 2708-2710．
23．糜涛．赵丽，郭小梅，等．tMfn2基因对大鼠血管平滑肌细胞凋亡的影响．临床心血管病杂志2007，24(9)：691-694．
24．Babapulle MN， Joseph L， Belisle P， et al．A hierarchical Bayesian meta-analysis of randomised clinical trials of drug-eluting stents．Lancet 2004， 364(9434)：583-591．
25．Liuzzo JP， Ambrose JA， Coppola JT．Sirolimus-and taxol-eluting stents differ towards intimal hyperplasia and re-endothelialization．J Invasive Cardiol 2005， 17(9)：497-502
1．Han DK， Haudenschild CC， Hong MK， et al．Evidence for apoptosis in human atherogenesis and in a rat vascular injury model．Am J Pathol 1995，147 (2)： 267-277．
2．Kockx MM， Knaapen MW．The role of apoptosis in vascular disease．J Pathol 2000，190(3)：267-280．
3．Guo XM， Chen KH， Guo YH， et al．Mitofusin 2 triggers vascular smooth muscle cell apoptosis via mitochondrial death pathway．Circ Res 2007， 101：1113-1122．
4．任澎，马东，马业新，等．p38MAPK信号途径与血管损伤后平滑肌细胞增殖关系的研究．临床心血管病杂志2005，21(12)：737-740．
5．Depree C， Taegtmeyer H．Metabolic aspects of programmed cell survival and cell death in the heart．Cardiovascular Res 2000， 45(3)：538-548．
6．Datta SR．Dudek H， Tao X， et al．Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery．Cell 1997， 91(2)： 231-241．
7．Cantley LC．The phosphoinositide3-Kinase pathway．Science 2002， 296 (5573)：1655-1657．
8．Helmut Lange， Harry Suryapranata， Giuseppe De Luca， et al， Folate therapy and in-stent restenosis after coronary stenting．N Engl J Med 2004．350(26)：2673-2681．
9．Rust C，Gores GJ．Apoptosis and liver disease．Am J Med 2000．108(7)：567-574．
10．Shimizu S，NaritaM，TsujimotoY．Bcl-2 family proteins regulate the release of apoptogenic cytochrome c by the mitochondrial channel VDAC．Nature 1999， 399(6735)：483-487．
11．Liston P．FongWG， Korneluk RG．The inhibitors of apoptosis： there is more to life than Bcl-2．Oncogene 2003， 22：8568-8580．
12．Chen KH， Guo X， Ma， et al．Dysregulation of HSG triggers vascular proliferative disorders．Nat Cell Biol 2004， 6(9)：872-883．
13．苗丽君．王静．Akt与肿瘤的研究进展．国外医学．生理、病理科学与临床分册 2004，24(5)：406-409．
14．Shen Tao， ZhenMing， Cao Chunmei， et al．Mitofusin-2 is a major determinant of oxidative stress-mediated heart muscle cell apoptosis．J Biol Chem 2007， 282(32)：23354-23361
15．Neuspiel M，Zunino R，Gangaraju S，et al．Activated mitofusin 2 signals mitochondrial fusion， interferes with Bax activation， and reduces susceptibility to radical induced depolarization．J Biol Chem 2005， 280(26)： 25060-25070．
16．Huang PW，Yu TZ，Yoon YS，et al．Mitochondrial clustering induced by overexpression of the mitochondrial fusion protein Mfn2 causes mitochondrial dysfunction and cell death．Euro J Cell Biol 2007， 86(6)：289-302．
17．廖华，曹文静，陈莉莉，等．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．中华急诊医学杂志2006， 15(6)：506-509．
18．刘亚萍，温绍君，刘雅，等．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．中华心血管病杂志2007，35(10)：914-918．
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2．Chen KH， Wang FQ， ZHANG J H， et al．Cloning and expression of a novel cDNA related to Hypertension．Chin Med J 1998， 111(4)：384-386．
3．Chen KH．Liu NK， Zhou AR， et al．The role of hypertension related gene in aorticvascular smooth muscle cells from mice and rats．Chin Med J 2001， 114(8)：833-836．
4．Hales KG and Fuller MT．Developmentally regulated mitochondrial fusion mediated by a conserved， novel， predicted GTPase．Cell 1997．90：121-129．
5．Hermann GJ，Thatcher JW， Mills JP，et al．Mitochondrial fusion in yeast requires the transmembrane GTPase Fzol．The Journal of Cell Biology 1998， 143：359-373．
6．Santel A and Fuller MT．Control of mitochondrial morphology by a human mitofusin．J Cell Sci 2001，114(5)：867-874．
7．Chen KH．Guo XM， Ma DL， et al．Dysregulation of HSG triggers vascular proliferative disorders．Nature Cell Biol 2004， 6(9)：872-883．
8．Santel A and Fuller MT．Control of mitochondrial morphology by a human Mitofusin．Journal of Cell Science 2000， 114：867-874．
9．Honda S．Aihara T， Hontani M， et al．Mutational analysis of action of mitochondrial fusion factor mitofusin-2．Journal of Cell Science 2005， 118：3153-3161．
10．Chen H， Detmer S， Andrew J， et al．Mitofusins Mfnl and Mfn2 coordinately regulate mitochondrial fusion and are essential for embryonic development．J Cell Biol 2003，160(2)：189-200．
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14．温绍君，王佐广，汤健等．Mfn2基因单核苷酸多态性与原发性高血压的相关性研究．中华预防医学杂志2005，39(1)：15-18．
15．Chien KR and Hoshijima M．Unravelling ras signals in cardiovascular disease．Nature Cell Biology 2004， 6：807-808．
16．Guo YH， Chen KH， Wei G， et al．Overexpression of Mitofusin 2 inhibited oxidized low-density lipoprotein induced vascular smooth muscle cell proliferation and reduced atherosclerotic lesion formation in rabbit．Biochemical and Biophysical Research Communications 2007， 363：411-417．
17．Guo XM， Chen KH， Guo YH， et al．Mitofusin 2 triggers vascular smooth muscle cell apoptosis via mitochondrial death pathway．Circulation Research 2007， 101：1113-1122．
18．Jiang GJ， Mei H， Bin Z， et al．Hyperplasia suppressor gene associates with smooth muscle a-actin and is involved in the redifferentiation of vascular smooth muscle cells．Heart Vessels 2006， 21：315-320．
19．Shen T， Zheng M， Cao CM， et al．Mitofusin-2 is a Major Determinant of Oxidative Stress-mediated Heart Muscle Cell Apoptosis．J Biol Chem 2007， 282 (32)：23354-23361．
20．Neuspiel M， Zunino R， Gangaraju S， et al．Activated mitofusin 2 signals mitochondrial fusion， interferes with Bax activation， and reduces susceptibility to radical induced depolarization．J Biol Chem 2005， 280： 25060-25070．
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1．Depre C．Taegtmeyer H．Metabolic aspects of programmed cell survival and cell death in the heart．Cardiovasc Res 2000，45(3)：538-548．
2．Westermann B．Merging mitochondria matters： Cellular role and molecular machinery of mitochondria fusion．EMBO Reports 2002，3(6)：527-531．
3．Chen H， Detmer SA，Ewald AJ，et al．Mitofusins Mfnl and Mfn2 coordinately regulate mitochondrial fusion and are essential for embryonic development．J Cell Biol 2003，160(2)：189-200．
4．Chen KH， Guo X， Ma D， et al．Dysregulation of HSG triggers vascular proliferation disorder．Nat Cell Biol 2004，6(9)：872-883．
5．Chien KR and Hoshijima M．Unravelling Ras signals in cardiovascular disease．Nat Cell Biol 2004，6：807-808．
6．Guo XM， Chen KH， Guo YH， et al．Mitofusin2 triggers vascular smooth muscle cell apoptosis via mitochondrial death pathway．Circ Res 2007，101(11)：1113-1122．
7， Chen KH， Wang FQ， Zhang JH， et al．Cloing and expression of a novel partial cDNA related to hypertension．Chin Med J 1998，111(4)：383-384．
8．Santel A， Fuller MT．Control of mitochondrial morphology by a human mitofusin．J Cell Sci 2001，114(5)：867-874．
9．Chen GH．Liu NK，Tang J，et al．The role of hypertension-related gene in aortic vascular smooth muscle cells from mice and rats．Chinese Medical Journal 2001，114(8)：833-836．
10．温绍君，王佐广，汤健等Mfn2基因单核苷酸多态性与原发性高血压的相关性研究．中华预防医学杂志2005，39(1)：15-18．
11．Jiang GJ，Mei H，Bin Z，et al．Hyperplasia suppressor gene associates with smooth muscle a-actin and is involved in the redifferentiation of vascular smooth muscle cells．Heart Vessels 2006，21：315-320．
12．Fang L， Moore XL， Gao XM， et al．Down-regulation of mitofusin-2 expression in cardiac hypertrophy in vitro and in vivo．Life Sciences 2007， 80： 2154-2160．
13．Chen CL．Shen T，Chen GH，et al．Inhibition of cardiac hypertrophy by mitofusin 2 in neonatal rat cardiomyocytes．Beijing Da Xue Xue Bao 2008， 40(5)：528-532．
14．Schaich M．lllmer T．Mdrl gene expression and mutations in Ras pro-To-oncogene in acute myeloid leukemia．Leuk Lymphoma 2002， 43：1345-1354．
15．Widmann C．Gibson S，Jarpe MB，et al．Mitogen-activated protein kinase：onservation of a three-kinase module from yeast to human．Pysiol Rev 1999，79(1)：143-180．
16．Garrington TP，Johnson GL．Organization and regulation of mitogen-activated protein kinase signaling pathway．Curr Opin Cell 1999，11(2)：211-218．
17．Melina PK．Timthy RB，Tzeng E．Gene therapy for restenosis．Circ Res 2000， 86：829-833．
18．Guo YH， Chen KH， GAO W， et al．Overexpression of Mitofusin 2 inhibited oxidized low-density lipoprotein induced vascular smooth muscle cell proliferation and reduced atherosclerotic lesion formation in rabbit．Biochem Biophys Res Commun 2007，363(2)：411-417
19．廖华，曹文静，陈莉莉，等．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中华急诊医学杂志2006，15(6)：506-509．
20．Datta SR， Dudek H， Tao X， et al．Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery．Cell 1997， 91(2)： 231-241．
21．Cantley LC．The phosphoinositide 3-Kinase pathway．Science 2002， 296(5573)： 1655-1657．
22．Lange H， Suryapranata H，De Luca G， et al．Folate therapy and in-stent restenosis after coronary stenting．N Engl J Med 2004， 350(26)： 2708-2710．
23．糜涛．赵丽，郭小梅，等．tMfn2基因对大鼠血管平滑肌细胞凋亡的影响．临床心血管病杂志2007，24(9)：691-694．
24．Babapulle MN， Joseph L， Belisle P， et al．A hierarchical Bayesian meta-analysis of randomised clinical trials of drug-eluting stents．Lancet 2004， 364(9434)：583-591．
25．Liuzzo JP， Ambrose JA， Coppola JT．Sirolimus-and taxol-eluting stents differ towards intimal hyperplasia and re-endothelialization．J Invasive Cardiol 2005， 17(9)：497-502
1．Han DK， Haudenschild CC， Hong MK， et al．Evidence for apoptosis in human atherogenesis and in a rat vascular injury model．Am J Pathol 1995，147 (2)： 267-277．
2．Kockx MM， Knaapen MW．The role of apoptosis in vascular disease．J Pathol 2000，190(3)：267-280．
3．Guo XM， Chen KH， Guo YH， et al．Mitofusin 2 triggers vascular smooth muscle cell apoptosis via mitochondrial death pathway．Circ Res 2007， 101：1113-1122．
4．任澎，马东，马业新，等．p38MAPK信号途径与血管损伤后平滑肌细胞增殖关系的研究．临床心血管病杂志2005，21(12)：737-740．
5．Depree C， Taegtmeyer H．Metabolic aspects of programmed cell survival and cell death in the heart．Cardiovascular Res 2000， 45(3)：538-548．
6．Datta SR．Dudek H， Tao X， et al．Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery．Cell 1997， 91(2)： 231-241．
7．Cantley LC．The phosphoinositide3-Kinase pathway．Science 2002， 296 (5573)：1655-1657．
8．Helmut Lange， Harry Suryapranata， Giuseppe De Luca， et al， Folate therapy and in-stent restenosis after coronary stenting．N Engl J Med 2004．350(26)：2673-2681．
9．Rust C，Gores GJ．Apoptosis and liver disease．Am J Med 2000．108(7)：567-574．
10．Shimizu S，NaritaM，TsujimotoY．Bcl-2 family proteins regulate the release of apoptogenic cytochrome c by the mitochondrial channel VDAC．Nature 1999， 399(6735)：483-487．
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