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

采用实时荧光定量PCR、免疫荧光染色、增殖实验、细胞凋亡实验和流式细胞术等方法探讨血管平滑肌细胞（vascular smooth muscle cell， VSMC）培养体系中分化、去分化和增殖的机制。大鼠引物200余组，犬引物10组，包括分化标记物SM1、SM2、SMemb、calponin、caldesmon等。利用反褶积软件和alexa荧光系列优化了非共聚焦显微镜的多色免疫染色成像方法，以获得更好的对比视图。利用这些工具，特别是实时PCR，我们评估了维生素C（AA）的作用，维生素C被认为可以调节VSMC增殖标志物。在10% FCS DMEM培养基中培养的VSMC中，AA增强SM1和钙钙蛋白的表达，但SM2的表达增强。由于SMemb在我们的环境中强烈表达，并不是所有的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下细胞和培养皿之间的界面。我们比较了VSMC培养在血浆处理的培养皿和明胶涂层表面。明胶包被增强了III型胶原蛋白、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的表达仅在血浆活化表面呈剂量效应增强，而在明胶涂层上没有。结果表明，在组织工程中，细胞与板或支架之间的界面可能在细胞分化和增殖中起重要作用。遗憾的是，使用质粒载体的基因转染试验仍在进行中，我们用质粒转染初级VSMC的效率约为30%。质粒转染原代细胞培养技术的进一步完善，将为VSMC的分化和去分化开辟新的研究领域。

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.