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Analysis of stabilization and degradation mechanism of muscle fiber of vascular smooth muscle cell upon transformatioon

血管平滑肌细胞转化后肌纤维的稳定和降解机制分析

基本信息

批准号：
15590224
负责人：
OKAGAKI Tsuyoshi
金额：
$ 2.3万
依托单位：
Mie University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

项目摘要

Cultured vascular smooth muscle cell line AC01 become to be in transformed state in the presence of PDGF, whereas it to be in well differentiated state in the presence of sodium butyrate (NaB). mRNA was isolated from AC01 cells cultured under these two types of conditions to make cDNA. We examined expression of several marker protein such as α-actin, high molecular weight caldesmon. By RT-PCR analysis, expression level of these two proteins was down regulated upon addition of PDGF, and up regulated in the presence of NaB. We further made subtraction library from these two types of cDNA as described below.To examine the nature of p32, a myosin regulatory protein, in living smooth muscle cell, we raised antibody against N-terminal signal sequence of the protein. By indirect immunofluorescence microscopy the antibody stained cytoskeletal filaments, suggesting the localization on myosin filaments. And western blotting using antibody against p32mat, that stain both p32mat and p32FL, showed that most of p32 existing in cytoskeleton of AC01 cell is p32FL (42 kDa) but not p32mat (38 kDa). These observations suggest that p32 is binding to myosin filament as p32FL containing signal sequence. Although p32mat bind to myosin and stimulate its assembly, p32FL expressed in E. coli did not interact with myosin. The discrepancy indicates that p32FL should be post-translationaly modified to interact with myosin in vivo.We further tried to make subtraction library to screen specific gene that is up regulated upon transformation. Using two types of cDNA that are obtained from PDGF treated and NaB treated cells, PCR-select subtraction have been performed to hybridize common gene in these two groups of cDNA. Subtracted cDNA was subcloned to plasmid to construct library.

培养的血管平滑肌细胞系AC 01在PDGF存在下变为转化状态，而在丁酸钠（NaB）存在下变为良好分化状态。从在这两种条件下培养的AC 01细胞中分离mRNA以制备cDNA。我们检测了几种标志蛋白的表达，例如α-肌动蛋白、高分子量钙调蛋白。通过RT-PCR分析，这两种蛋白的表达水平在加入PDGF后下调，在NaB存在下上调。为了研究活平滑肌细胞中肌球蛋白调节蛋白p32的性质，我们制备了针对该蛋白N-末端信号序列的抗体。通过间接免疫荧光显微镜，抗体染色细胞骨架丝，表明肌球蛋白丝的定位。用p32 mat抗体进行蛋白质印迹分析，结果显示AC 01细胞骨架中p32主要以p32 FL（42 kDa）存在，而p32 mat（38 kDa）不存在。提示p32以p32 FL信号序列的形式与肌球蛋白丝结合。虽然p32 mat与肌球蛋白结合并刺激其组装，但p32 FL在E.大肠杆菌不与肌球蛋白相互作用。我们进一步尝试构建差减文库，筛选在转化过程中表达上调的基因。用PDGF处理的和NaB处理的两组cDNA，进行PCR选择消减杂交，以杂交这两组cDNA中的共同基因。亚克隆到质粒上构建文库。