中华急诊医学杂志
中華急診醫學雜誌
중화급진의학잡지
CHINESE JOURNAL OF EMERGENCY MEDICINE
2009年
8期
805-809
,共5页
赵丽%王四坤%周炜%廖华%张文娟%陈光慧%郭小梅
趙麗%王四坤%週煒%廖華%張文娟%陳光慧%郭小梅
조려%왕사곤%주위%료화%장문연%진광혜%곽소매
线粒体融合素基因2%细胞增殖%腺病毒%血管平滑肌细胞
線粒體融閤素基因2%細胞增殖%腺病毒%血管平滑肌細胞
선립체융합소기인2%세포증식%선병독%혈관평활기세포
Mitofusin2%Cell proliferation%Adenovirus%Vascular smooth muscle cell
目的 比较线粒体融合素基因2(Mfn2)和去除穿膜区序列的大鼠线粒体融合素基因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.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;流式细胞术检测tMfn2基因对VSMCs周期的影响;Western blot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.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);流式细胞术结果表明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基因对细胞剧期的影响更明显(P<0.01).进一步研究表明tMfn2比Mfn2更能降低磷酸化ERK1/2(p-ERK1/2)和磷酸化Raf-1(p-Raf-1)的表达水平(P<0.01).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.
目的 比較線粒體融閤素基因2(Mfn2)和去除穿膜區序列的大鼠線粒體融閤素基因2(tMfn2)對大鼠血管平滑肌細胞(VSMCs)增殖的作用,探討tMfn2基因對VSMCs增殖的影響及其相關的信號通路.方法 用攜帶tMfn2基因和Mfn2基因的重組腺病毒(Adv-tMfn2和Adv-Mfn2)感染VSMCs,檢測tMfn2蛋白和Mfn2蛋白在細胞中的錶達水平;細胞計數法、四甲基偶氮唑鹽(MTT)法檢測VSMCs的增殖;流式細胞術檢測tMfn2基因對VSMCs週期的影響;Western blot分析各組燐痠化ERK1/2和燐痠化Raf-1蛋白錶達水平的變化;採用方差分析對數據進行統計學處理.結果 Adv-tMfn2和Adv-Mfn2感染VSMCs後能有效錶達齣相應蛋白;細胞計數和MTT結果顯示,tMfn2和Mfn2均使VSMCs增殖受到明顯抑製(P<0.01),且前者較後者抑製效果更明顯(P<0.01);流式細胞術結果錶明tMfn2和Mfn2使多數VSMCs停滯于G0/G1期,細胞比例為(88.01±4.38)%和(67.43±6.21)%,可見tMfn2基因對細胞劇期的影響更明顯(P<0.01).進一步研究錶明tMfn2比Mfn2更能降低燐痠化ERK1/2(p-ERK1/2)和燐痠化Raf-1(p-Raf-1)的錶達水平(P<0.01).結論 與Mfn2基因相比,tMfn2基因更能顯著抑製VSMCs增殖,使細胞週期停滯于G0/G1期.這一作用主要通過抑製Ras-Raf-ERK1/2信號通路,下調燐痠化Raf-1蛋白錶達,進而抑製ERK1/2的燐痠化而實現.
목적 비교선립체융합소기인2(Mfn2)화거제천막구서렬적대서선립체융합소기인2(tMfn2)대대서혈관평활기세포(VSMCs)증식적작용,탐토tMfn2기인대VSMCs증식적영향급기상관적신호통로.방법 용휴대tMfn2기인화Mfn2기인적중조선병독(Adv-tMfn2화Adv-Mfn2)감염VSMCs,검측tMfn2단백화Mfn2단백재세포중적표체수평;세포계수법、사갑기우담서염(MTT)법검측VSMCs적증식;류식세포술검측tMfn2기인대VSMCs주기적영향;Western blot분석각조린산화ERK1/2화린산화Raf-1단백표체수평적변화;채용방차분석대수거진행통계학처리.결과 Adv-tMfn2화Adv-Mfn2감염VSMCs후능유효표체출상응단백;세포계수화MTT결과현시,tMfn2화Mfn2균사VSMCs증식수도명현억제(P<0.01),차전자교후자억제효과경명현(P<0.01);류식세포술결과표명tMfn2화Mfn2사다수VSMCs정체우G0/G1기,세포비례위(88.01±4.38)%화(67.43±6.21)%,가견tMfn2기인대세포극기적영향경명현(P<0.01).진일보연구표명tMfn2비Mfn2경능강저린산화ERK1/2(p-ERK1/2)화린산화Raf-1(p-Raf-1)적표체수평(P<0.01).결론 여Mfn2기인상비,tMfn2기인경능현저억제VSMCs증식,사세포주기정체우G0/G1기.저일작용주요통과억제Ras-Raf-ERK1/2신호통로,하조린산화Raf-1단백표체,진이억제ERK1/2적린산화이실현.
Objective To investigate the effect of tMfn2 gene on inhibiting the proliferation of vascular smooth muscle cells (VSMCs) and related mechanism. Method VSMCs were transfected with adenovirus vector encoding tMfn2 or Mfn2 (Adv-tMfn2, Adv-Mfn2). The abundance of tMfn2 protein and Mfn2 protein were deter-mined by Western blot analysis using Mfn2 N-term antibody. The effect of tMfn2 on the proliferation of VSMCs was explored by cell counting and MTT assay. The cell cycle was analyzed using flow cytometry. Western blot were used to detect the expression of p-ERK1/2 and p-Raf-1. Results The results of cell counting and MTT both indi-cated that tMfn2 gene displayed more remarkable effect on inhibiting the proliferation of VSMCs than Mfn2 (P <0.01). Flow cytometry showed that most of the cells infected with Adv-tMfn2 or Adv-Mfn2 were blocked in the stage of G0/G1 and few entered into the S phase. Western blot indicated that overexpression of tMfn2 gene resulted in downregulation of phosphorylated ERK1/2 and Raf-1 protein (P < 0.01). These results demonstrated tMfn2 had stronger effect than Mfn2 (P < 0.01). Conclusions tMfn2 gene is superior to Mfn2 gene in attenuating the proliferation of VSMCs via the Ras-Raf-ERK1/2 signaling pathway.
