CAJ | 학술논문

目的：研究线粒体融合蛋白基因2（Mfn-2）降低表达对培养的大鼠血管平滑肌细胞（rVSMCs）中线粒体自噬的影响。方法将 rVSMCs 采用无血清培养基同步化48 h 后，分别感染腺病毒载体 Adv-Mfn-2-SiRNA 和空载 Adv-Lac-Z 作为实验组和对照组，实验分为三部分，其中第一部分提取细胞总蛋白后进行 Western Blot 分析 Mfn-2的表达，第二部分 JC-1处理后运用流式细胞术检测线粒体膜电位的变化，第三部分通过透射电子显微镜显示线粒体超微结构的改变以及自噬体的形成和积累过程。结果 Adv-Mfn-2-SiRNA 以感染复数60pfu/细胞感染同步化后的 rVSMCs 24 h 后，Western Blot 分析表明， Mfn-2的表达与对照组相比明显降低（P ＜0.05）；流式细胞术检测结果显示实验组较对照组线粒体膜电位明显下降（P ＜0.05）；透射电子显微镜显示实验组线粒体减少，而自噬体明显增多。结论 Mfn-2降低可导致培养的大鼠血管平滑肌线粒体自噬大量增加。
목적：연구선립체융합단백기인2（Mfn-2）강저표체대배양적대서혈관평활기세포（rVSMCs）중선립체자서적영향。방법장 rVSMCs 채용무혈청배양기동보화48 h 후，분별감염선병독재체 Adv-Mfn-2-SiRNA 화공재 Adv-Lac-Z 작위실험조화대조조，실험분위삼부분，기중제일부분제취세포총단백후진행 Western Blot 분석 Mfn-2적표체，제이부분 JC-1처리후운용류식세포술검측선립체막전위적변화，제삼부분통과투사전자현미경현시선립체초미결구적개변이급자서체적형성화적루과정。결과 Adv-Mfn-2-SiRNA 이감염복수60pfu/세포감염동보화후적 rVSMCs 24 h 후，Western Blot 분석표명， Mfn-2적표체여대조조상비명현강저（P ＜0.05）；류식세포술검측결과현시실험조교대조조선립체막전위명현하강（P ＜0.05）；투사전자현미경현시실험조선립체감소，이자서체명현증다。결론 Mfn-2강저가도치배양적대서혈관평활기선립체자서대량증가。
Objective To explore whether Mfn-2 could play some role in the generation of mitochon-dria autophagy in cultured rat vascular smooth muscle cells (rVSMCs).Methods Adenoviral infection was carried out by adding 60 pfu cell-h. Adv-LacZ served as a control. Then three experiments were performed. Firstly ,the proteins from two groups were extracted for Western Blot to detect Mfn-2 expression in cells. Secondly ,two groups’ cells were analyzed with MitoProbeT M JC-1 assay. And thirdly ,cells were observed with transmission electron microscopy. Results Western Blot demonstrated that the expression of Mfn-2 significantly decreased in rVSMCs with adenoviral infection of Adv-Mfn-2-SiRNA. Mitochondrial membrane potential detected by MitoProbeT M JC-1 assay significantly decreased in the Adv-Mfn-2-SiRNA infection group. Using transmis-sion electron microscopy ,we confirmed that there were mitochondria decrease and autophagosome accu-mulation in the group with adenoviral infection of Adv-Mfn-2-SiRNA. Conclusion Depression of Mfn-2 promotes the accumulation of autophagosomes in the vascular smooth muscle cells.