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

培养的血管平滑肌细胞系AC01在PDGF的作用下呈转化状态，而在丁酸钠（NaB）的作用下呈分化状态。从这两种条件下培养的AC01细胞中分离mRNA，制备cDNA。我们检测了α-肌动蛋白、高分子量caldesmon等标记蛋白的表达。通过RT-PCR分析，这两种蛋白的表达水平在添加PDGF时下调，在NaB存在时上调。我们进一步从这两种类型的cDNA构建如下所述的减法文库。为了研究肌球蛋白调节蛋白p32在活的平滑肌细胞中的性质，我们制备了针对该蛋白n端信号序列的抗体。间接免疫荧光显微镜下，抗体染色细胞骨架丝，提示定位于肌球蛋白丝。p32mat抗体western blotting显示，AC01细胞骨架中存在的p32大部分是p32FL (42 kDa)，而不是p32mat （38 kDa）。这些观察结果表明p32作为含有信号序列的p32FL与肌球蛋白丝结合。虽然p32mat与肌凝蛋白结合并刺激其组装，但在大肠杆菌中表达的p32FL不与肌凝蛋白相互作用。这种差异表明p32FL在体内应该经过翻译后修饰才能与肌球蛋白相互作用。我们进一步尝试制作减法文库来筛选在转化过程中上调的特定基因。利用从PDGF处理和NaB处理的细胞中获得的两种类型的cDNA，对这两组cDNA中的共同基因进行pcr选择减法杂交。将缺失的cDNA亚克隆到质粒上构建文库。

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.