Mfn-2对rVSMCs细胞周期的调控作用

【作者】 李丹；

【导师】 郭小梅；

【作者基本信息】 华中科技大学 ， 分子心脏病学， 2013， 博士

【摘要】 第一部分Mfn-1和Mfn-2在rVSMCs细胞周期中的表达差异研究目的：研究线粒体融合素基因-2与线粒体融合素基因-1在大鼠血管平滑肌细胞周期中的表达差异,旨在阐明对大鼠血管平滑肌细胞周期具有调控作用的线粒体融合素基因。方法：采用无血清培养基同步化24小时,无血清培养基同步化48小时后换用含10%血清的培养基再生长18小时,胸腺嘧啶核苷和诺考达唑先后作用12小时诱导大鼠血管平滑肌细胞进入细胞周期不同阶段,分为两组,其中一组PI染色后进行流式细胞术检测,另一组提取细胞总蛋白后进行Western Blot分析线粒体融合素基因-2、线粒体融合素基因-1和细胞周期依赖性激酶抑制剂Ip27、p21、pRb的表达。结果：流式细胞术结果显示,无血清培养基同步化24小时后,平均80.10%的大鼠血管平滑肌细胞进入Go/Gl期；无血清培养基同步化48小时后换用含10%血清的培养基再生长18小时后,平均61.27%的大鼠血管平滑肌细胞进入S期；胸腺嘧啶核苷和诺考达唑先后作用12小时后,平均75.06%的大鼠血管平滑肌细胞进入G2/M期；处于正常生长周期中的大鼠血管平滑肌细胞Go/G1期、S期和G2/M期的细胞分布平均为51.36%,39.52%,9.12%,作为对照。Western Blot分析表明,Go/G1期线粒体融合素基因-2和细胞周期依赖性激酶抑制剂p27、p21的表达与对照组和G2/M期相比明显升高(P<0.05), pRb的表达降低；而线粒体融合素基因-1G0/G1期的表达与对照组相比,无明显差异。结论：在大鼠血管平滑肌细胞G0/G1期线粒体融合素基因-2的表达明显增高,而线粒体融合素基因-1的表达并无明显变化,表明线粒体融合素基因-2对大鼠血管平滑肌的细胞周期具有调控作用。第二部分rVSMCs细胞周期不同阶段形态与代谢的变化研究目的：研究大鼠血管平滑肌细胞周期不同阶段细胞器的形态改变和代谢变化。方法：将大鼠血管平滑肌细胞阻滞于细胞周期的不同阶段,采用激光共聚焦显微镜观察经MitoTracker Red染色的线粒体形态的变化；通过透射电子显微镜显示线粒体超微结构的改变、线粒体与内质网毗邻关系的变化以及自噬溶酶体的形成和积累过程；JC-1处理后运用流式细胞术检测线粒体膜电位的变化；采用化学法葡萄糖检测试剂盒、氨基酸检测试剂盒、ATP比色测定试剂盒、NAD+/NADH定量检测试剂盒和NADP+/NADPH定量检测试剂盒检测葡萄糖代谢、氨基酸代谢、ATP代谢、NAD+/NADH和NADP+/NADPH的变化。结果：激光共聚焦显微镜观察结果显示,Go/G1期线粒体呈管网状分布,而对照组以及S期线粒体呈散在的粒状分布,G2/M期线粒体呈散在的团状分布；进一步通过透射电子显微镜显示Go/G1期线粒体与内质网的毗邻关系更近,而且内质网甚至被拉向线粒体；S期线粒体与内质网分离,而且线粒体肿胀、嵴断裂,内质网池扩张；G2/M期线粒体数量减少,出现大量的自噬溶酶体。流式细胞术检测表明,从Go/G1期至G2/M期线粒体膜电位明显下降(P<0.05)。采用化学法检测发现,葡萄糖代谢、氨基酸代谢、ATP代谢和NADP+/NADPH在整个细胞周期中并未明显变化,NAD+含量在Go/G1期明显降低(P<0.05)。结论：尽管在大鼠血管平滑肌细胞的整个细胞周期中,葡萄糖、氨基酸和ATP代谢无显著改变,但是在大鼠血管平滑肌细胞Go/G1期,当线粒体融合素基因-2表达升高时,线粒体密集呈管网状分布,且内质网被拉向线粒体,NAD+含量明显降低；而在大鼠血管平滑肌细胞G2/M期,当线粒体融合素基因-2含量降低时,线粒体膜电位急剧下降,伴随大量的自噬溶酶体形成。第三部分Mfn-2对rVSMCs线粒体重塑的影响[目的]研究过表达线粒体融合素基因-2对大鼠血管平滑肌细胞线粒体形态变化的影响。[方法]构建Mfn-2的腺病毒载体Adv-Mfn-2-S442A,采用Adv-Mfn-2-S442A的不同感染复数25pfu/细胞和50pfu/细胞感染血管平滑肌细胞24小时后,激光共聚集显微镜观察MitoTracker Red染色后线粒体形态的变化。[结果]成功构建Mfn-2的腺病毒载体Adv-Mfn-2-S442A; Adv-Mfn-2-S442A以感染复数25pfu/细胞感染血管平滑肌细胞24小时后,激光共聚集显微镜显示MitoTracker Red染色的线粒体在细胞浆内呈散在团状分布；Adv-Mfn-2-S442A以不同感染复数50pfu/细胞感染血管平滑肌细胞24小时后,激光共聚集显微镜显示MitoTracker Red染色的线粒体在细胞质内呈网状分布,且出现明显的核周聚集。[结论]过表达Mfn-2可促使血管平滑肌细胞的线粒体形成网状结构。更多还原

【Abstract】 Part One:Difference between Mfn-1and Mfn-2Expression in the Cell Cycle of Vascular Smooth Muscle Cells[Aims] To explore whether Mfn-2or Mfn-1could play some roles in the regulation of cell cycle, we investigated the endogenous expressional difference between Mfn-2and Mfn-1in the cell cycle of vascular smooth muscle cells.[Methods] Cell cycle arrest was achieved by24h of serum deprivation,18h of serum culture after48h of serum deprivation and treatment of nocodazole for12h after thimidine for12h. Cells induced to the same phase were divided into two groups, one for analysis by propidium iodide staining and the other for Western Blot.[Results] Flow cytometry analysis showed the cells were arrested to Go/Gi, S and G2/M phase of the cell cycle of the vascular smooth muscle cells. The untreated cells with70%confluence were used as controls, indicating the average distributions of G0/G1, S and G2/M phase51.36%,39.52%and9.12%respectively. Western Blotting demonstrated more pronounced expression of endogenous Mfn-2, p27and p21and decrease of pRb in the G0/G1phase of the vascular smooth muscle cells, while no change of the expression of endogenous Mfh-1in the G0/G1phase.[Conclusion] G0/G1cell cycle arrest induced the upregulation of Mfn-2not Mfn-1, suggesting a regulatory role of Mfn-2on the cell cycle progression of vascular smooth muscle cells. Part Two:Morphological and Metabolic Changes in the Cell Cycle of Vascular Smooth Muscle Cells[Aims] To study the relationship between the morphology and function of Mfn-2, we examined the morphological and metabolic changes in the cell cycle of vascular smooth muscle cells.[Methods] MitoTracker Red was stained in the vascular smooth muscle cells induced into the different phase of the cell cycle to show the mitochondrial morphology by confocal fluorescence microscopy. Using transmission electron microscopy, we observed the ultrastructure variation during the cell cycle of vascular smooth muscle cells. Mitochondrial membrane potential was detected by MitoProbeTM JC-1assay and the energization alteration by QuantiChrom Glucose Assay Kit, L-Amino Acid Quantitation Kit, ATP colorimetric assay and NAD(P)+/NAD(P)H measurements.[Results] Laser confocal fluorescence microscopy showed that the mitochondria were in a tubular network state in the Go/G1phase but in a fragmented solitary spheroid state in the S phase and in a separated group state in the G2/M phase. Using transmission electron microscopy, we confirmed that there were many more mitochondria present and the endoplasmic reticulum (ER) was tethered to the mitochondria in the G0/G1phase, swelling mitochondria with destructed cristae in the S phase and autophagosome accumulation in the G2/M phase. Although glucose measurement, L-Amino Acid Quantitation and ATP production remained unchanged during the cell cycle progression, NAD+levels were lower in the G0/G1phase and mitochondrial membrane potential decreased in the G2/M phase.[Conclusion] The energization has no change during the cell cycle of vascular smooth muscle cells. However, mitochondria were in a tubular network state and endoplasmic reticulum was tethered to mitochondria when Mfn-2was upregulated in the G0/G1phase. Furthermore, mitochondria were in a fragmented solitary spheroid state and autophagosomes were accumulated with decreased mitochondrial membrane potential when Mfn-2was downregulated in the S and G2/M phase. Part Three:Effect of Overexpression of Mfn-2On the Mitochondrial remodeling in the Vascular Smooth Muscle Cells[Aims] To investigate the effect of Mfn-2on mitochondrial remodeling, we applied Adenovirus vector of Mfn-2in the vascular smooth muscle cells.[Methods] Adv-Mfn-2-S442A was constructed as previously. Adenoviral infection was carried out by adding25pfu cell-1and50pfu cell-1Adv-Mfn-2-S442A into the vascular smooth muscle cells after synchronized for48h. Adv-LacZ was served as a control. Then the treated cells were stained with MitoTracker Red and observed by laser confocal fluorescence microscopy.[Results] Adenovirus vector Adv-Mfn-2-S442A was successfully constructed. Using laser confocal fluorescence microscopy, the vascular smooth muscle cells stained with MitoTracker Red after the treatment of adenoviral infection displayed a solitary group gathering distribution when infected with25pfu cell-1of Adv-Mfn-2-S442A and a reticulum state with perinuclear clustering with50pfu cell-1of Adv-Mfn-2-S442A. [Conclusion] Overexpression of Mfn-2promotes the mitochondria to be in a tubular network state in the vascular smooth muscle cells.更多还原