| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2005: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 2004: ¥2,400,000 (Direct Cost: ¥2,400,000)
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| Research Abstract |
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.
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