起始识别复合物1基因在血管平滑肌细胞DNA复制中的作用

目的探讨起始识别复合物1（ORC1）在血管平滑肌细胞（VSMC）DNA复制中的表达及其意义。方法采用组织块贴片法原代培养的大鼠胸主动脉VSMC,利用双胸苷阻断、秋水仙素阻抑法和血清饥饿法使VSMC达到细胞周期同步化,用逆转录聚合酶链反应（RT-PCR）技术和Western blot检测不同细胞周期的VSMC ORC1 mRNA和蛋白表达。结果同步化的VSMC DNA含量百分比,血清饥饿法以G0G1期为主（P〈0．01）,双胸苷阻断14h以G0G0期为主（P〈0．01）,双胸苷阻断14h后10％胎牛血清刺激6h以S期为主（P〈0．01）,秋水仙素培养12h以G2／M期为主（P〈0．05）。静止状态VSMC的ORC1 mRNA和蛋白质无表达,处于G1／S期VSMC的ORC1 mRNA表达量显著高于S期和G2／M期。VSMC的ORC1蛋白质表达量与ORC1 mRNA变化规律相似。结论ORC1随细胞的分裂周期而表达,ORC1可能是启动VSMC DNA复制的关键因子。

Expression of origin recognition complex 1 gene in DNA replication of vascularsmooth muscle cells

OBJECTIVE: To explore the expression of origin recognition complex 1 (ORC1) during the DNA replication of vascular muscle cells (VSMC). METHODS: VSMC of thoracic aorta in rats were obtained by the adherence method of tissue culture. The cell synchrony was obtained by the method of double-thymidine block, colchicine treatment and serum starvation. The expression of ORC1 mRNA at different cell cycles of VSMC was determined by RT-PCR and the protein expression of ORC1 was analyzed by Western blot. RESULTS: Cultured VSMC were identified by light microscope and immunocytochemistry. Significant expression of ORC1 mRNA and protein in a quiescent stage of VSMC were not observed. Upon synchronization, the expression of ORC1 mRNA was significantly higher at G(1)/S phase of VSMC than that at S and G(2)/M phases. The expression of ORC1 protein followed same changes as the ORC1 mRNA expression at different stages of cell cycles. CONCLUSION: ORC1 may be an important regulatory factor at the initiation of proliferative process of VSMC.