CTGF反义RNA对血管平滑肌细胞增殖和迁移的影响.doc

CTGF反义RNA对血管平滑肌细胞增殖和迁移的影响
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【摘要】目的应用反义RNA技术特异性下调结缔组织生长因子(CTGF)的表达,观察其对血管平滑肌细胞(VSMC)增殖和迁移的影响,并探讨其作用机制。方法培养原代VSMC,(+)-CTGF(-)转染入VSMC,Western Blot技术观察CTGF的表达以及蛋白激酶B(Akt)的活性,MTT法及划痕试验测定VSMC增殖和迁移情况。结果 (+)-CTGF(-)转染VSMC后CTGF的表达明显降低,Akt的活性降低,VSMC的增殖和迁移受到明显抑制。结论应用反义RNA技术可特异性下调CTGF表达,并通过降低Akt的活性抑制VSMC的增殖和迁移。
【关键词】血管平滑肌细胞结缔组织生长因子细胞增殖细胞迁移反义RNA
Abstract:Objective To specifically down-regulate the expression of CTGF by applying anti-sense RNA technique and observe its effect on proliferation and migration of VSMC. Methods Primary VSMCs were cultured by adherence method. Anti-sense RNA technique was applied to specifically down-regulate the expression of CTGF. Expression of CTGF and activity of Akt were detected by WB. Proliferation and migration of VSMC were determined by MTT and scratch assay. Results Transfer of pcDNA (+)-CTGF(-) could specifically down-regulate CTGF expression and the activity of Akt. The proliferation and migration of VSMC were inhibited. Conclusions Specific down-regulation of CTGF expression by anti-sense RNA technique could inhibit the proliferation and migration of VSMCs by decreasing the phosphorylation of Akt.
Key words:vascular Smooth muscle cell (VSMC);connective tissue growth factor (CTGF);cellproliferation;cell migration;anti-sense RNA
动脉粥样硬化(Atherosclerosis,AS)始于血管内膜损伤[1]。在AS发生前,常有多种因素的刺激对血管内膜造成损伤,内皮细胞发生功能性和器质性改变,造成通透性改变和分泌功能障碍,促进血液中的脂质进入动脉壁。内皮细胞和动脉中层的血管平滑肌细胞(vascular endothelial cell,VSMC)进一步激活,大量合成并分泌多种生长因子,使自身和周围细胞大量增殖,增殖的VSMC还可迅速合成胶原等细胞外基质[2],促使VSMC在损伤修复过程中发生纤维化,形成瘢痕修复,血管壁在结构和功能上发生了变化,最终导致了AS的形成[3]。
结缔组织生长因子(connective tissue growth factor,CTGF)是从培养的人脐带静脉血内皮细胞的条件培养基中发现的。有研究表明[4],CTGF参与了血管的损伤修复,能够直接诱导结缔组织细胞增殖和细胞外基质合成,促进AS的发展。AS病灶中CTGF的mRNA和蛋白质较正常的血管内有较高表达,可见CTGF在AS的发生和发展中有着重要意义[5]。
本课题旨在探讨CTGF在AS中对VSMC增殖和迁移的影响并明确其相关机制,为CTGF对动脉粥样硬化性血管狭窄病变过程的影响提供实验依据。
1 材料与方法
实验材料
1月龄SD大鼠由辽宁医学院实验动物中心提供。(+)-CTGF(-)质粒由辽宁医学院科学实验中心