Screening of genes those are related to dynamic transition of myofiber of vascular smooth muscle cell upon phenotypic modulation.

血管平滑肌细胞在不同情况下表现为收缩型和合成型两种典型表型。我们利用培养的VSMC， P53LMAC01建立了表型调节模型系统。添加PDGF后，VSMC转变为合成型表型，在丁酸钠（Na-B）存在下，VSMC转变为典型的收缩型表型，丁酸钠通过抑制DNA的乙酰化诱导生长抑制。从这两种典型细胞中分离mRNA，构建减法cDNA文库。我们对文库的插入片段进行测序，从pdgf特异性文库中筛选出230个克隆，从nab特异性文库中筛选出160个克隆。将每个克隆的进一步插入点定位到尼龙膜上，并通过假定的Northern杂交检查以确认每种类型细胞的特异性。将四组膜与四组探针、两个从pdgf和nab处理的细胞中减去的cdna和两个原始cdna进行杂交。结果证实pdgf特异性文库中的15个克隆和nab特异性文库中的19个克隆的表达具有特异性。为了量化这些克隆在pdgf和nab处理的细胞中的表达水平，我们为每个克隆制作了引物集，并用real - time PCR检测。s100a4、S100A6、α-actin等克隆在两种细胞间表达差异为2倍。采用RT-PCR方法分离出S 100A4和S 100A6的全长cDNA，并在细菌中表达。利用这些基因产物，我们从VSMC中亲和纯化了3个与s1004和S100A6蛋白相互作用的蛋白。

Vascular smooth muscle cell (VSMC) shows two typical phenotype, contraction-type and synthetic-type upon various situations. We developed model system of phenotypic modulation by using cultured VSMC, P53LMAC01. Upon addition of PDGF, the VSMC changed to synthetic-type phenotype, and it changed to typical contraction-type phenotype in the presence of sodium butyrate (Na-B), which induce growth suppression by inhibiting acetylation of DNA. We isolated mRNA from these typical two types of cell and constructed subtraction cDNA library. We sequenced inserts of the library, and screened 230 clones from PDGF-specific library, and 160 clones from NaB-specific library. Further inserts of each clone were spotted to nylon membrane and examined by putative Northern hybridization to confirm the specificity in each types of cell. Four sets of membrane was hybridized with four sets of probes, two subtracted cDNAs and two original cDNAs from PDGF-and NaB-treated cells. As a result, 15 clones from PDGF-specific library and 19 clones from NaB-specific library were confirmed in the specificity of the expression. To quantify the expression level of these clones in PDGF-and NaB-treated cell, we made primer sets for each clone and examined with real time PCR. Clones showing 2-fold difference of expression among these two types of cell were S 100A4, S100A6, α-actin and others. We isolated the full-length cDNA of S 100A4 and S 100A6 by RT-PCR, and expressed these genes in bacteria. Using these gene products we affinity purified three proteins that interact with S 1004 and S100A6 proteins from the VSMC.