Control of differentiation, dedifferentiation and proliferation by modulation of intracellular signal cascades in vascular smooth muscle cell.

本研究采用实时荧光定量PCR、免疫荧光染色、细胞增殖实验、细胞凋亡实验和流式细胞术等技术，探讨血管平滑肌细胞（VSMC）在体外培养体系中的分化、去分化和增殖机制。优化了200多对大鼠引物和10对犬引物，包括SM1、SM2、SMemb、calponin、caldesmon等分化标志物。使用非共聚焦显微镜进行多色免疫染色成像的方法也进行了优化，以使用去卷积软件和alexa荧光系列获得更好的对比度视图。使用这些工具，特别是使用实时PCR，我们评估了维生素C（AA）的作用，其被认为调节VSMC增殖标记物。AA促进10%FCS DMEM培养的VSMC表达SM1和calponin，但SM2表达增加。由于SMemb在我们的环境中强烈表达，并非所有增殖标志物都通过维生素C刺激表达。另一方面，我们评估了不同浓度维生素C下细胞和平板之间的界面。我们比较了在等离子体处理的平板上或在明胶涂覆的表面上的VSMC培养物。明胶涂层增强了III型胶原、整合素β 1和整合素α 5的表达。有趣的是，随着维生素C浓度的增加，弹性蛋白mRNA的表达以剂量响应方式在等离子体活化的表面上增强，而不是在明胶涂层上。结果表明，细胞与组织工程中的板或支架之间的界面对细胞的分化和增殖可能起重要作用。遗憾的是，利用质粒载体的基因转染试验仍在进行中，我们用质粒对原代VSMC的转染效率约为30%。进一步完善原代培养细胞的质粒转染技术，将为VSMC的分化和去分化研究开辟新的领域。

We developed real-time PCR, immunofluorescent staining, proliferation assay, apoptosis assay, and flow cytometry to explore a mechanism of differentiation, dedifferentiation, and proliferation in vascular smooth muscle cell (VSMC) culture system. Primer sets more than 200 in the rats and 10 in the dog were optimized including differentiation markers, such as SM1, SM2, SMemb, calponin, caldesmon. Methods of multi-color immunostaining imaging with a non-confocal microscope also optimized to get better contrast views using deconvolution software and alexa fluorescent series. With these tools, especially with real-time PCR, we assessed an effect of Vitamin C(AA), which has considered to modulate VSMC proliferation markers. AA enhanced expression of SM1 and calponin, but SM2 in VSMC cultured in 10% FCS DMEM medium. Since SMemb was strongly expressed in our settings, not all proliferation markers was expressed by Vitamin C stimulation. On the other hand, we assessed interfaces between cells and plates in the various concentration of Vitamin C. We compared VSMC cultures on plasma treated plates or on gelatin coated surface. Gelatin coating enhanced expressions of collagen type III, Integrin beta1, and integrin alpha5. Interestingly, as concentration of vitamin C is increased, elastin mRNA expression was enhanced in a dose response manner junst on the plasma activated surface, not on gelatin coating. The result suggests that interfaces between cells and plates, or scaffold in tissue engineering may play an important roll in cell differentiation and proliferation. Unfortunately, gene transfection trial using plasmid vector is still on process, we achieved about 30% transfection efficiency for primary VSMC with plasmids. More refinement of transfection technique on primary cell culture using plasmid may develop new fields of research in differentiation and dedifferentiation of VSMC.