细胞周期蛋白A对培养大鼠血管平滑肌细胞起始识别复合物1表达水平的影响

目的 探讨细胞周期蛋白A （Cyclin A）对培养大鼠血管平滑肌细胞（VSMCs）周期起始识别复合物1（ORC1）表达水平的影响.方法 采用组织块贴片法原代培养的大鼠胸主动脉血管平滑肌细胞,利用双胸苷使VSMCs达到细胞周期同步化,应用逆转录聚合酶链反应技术和流式细胞仪检测不同细胞周期VSMCs的ORC1和Cyclin A mRNA和蛋白表达.结果 经鉴定培养的细胞为VSMCs,采用血清饥饿法、双胸苷阻断法和秋水仙素阻抑法分别获得了处于不同细胞周期的同步化的VSMCs：G0/G1期（89.22 ±3.54）％,G1/S交界期（66.74±7.16）％,S期（63.24±4.06）％,G2/M期（51.64±11.18）％,各期比较差异有统计学意义（P均＜0.01）.Cyclin A在静止状态对VSMCs的ORC1 mRNA和蛋白无影响,在G2/M期达到高峰[（52.133 3 ±2.122 1）％],与G1/S期[（10.9167±0.531 1）％]、S期[（7.656 7±0.412 4）％]比较,差异有统计学意义（P均＜0.01）.ORC1在G2/M期达到最低值[（1.276 7 ±0.161 7）％],与G1/S期[（13.371 0±1.057 3）％]、S期[（3.043 3 ±0.538 0）％]比较,差异有统计学意义（P均＜0.01）,可能原因是Cyclin A阻止ORC1结合在VSMCs染色质上.结论 CyclinA可能调控ORC1在VSMCs细胞周期的启动.

Effect of Cyclin A on origin recognition complex-1 expression of rat vascular smooth cells

Objective To explore the effect of Cyclin A on origin recognition complex-1 （ORC1） expression of rat vascular smooth cells （VSMCs）.Methods Primary VSMCs of thoracic aorta in rats was obtained by the adherence method of tissue culture.The synchrony of cell was obtained by the method of double-thymidine block.In different cell cycles of VSMCs,the expression of ORC1 mRNA was determined by RT-PCR and the protein expression of ORC1 was observed by flow cytometry.Results Synchronized VSMCs were obtained and identified by the methods of double-thymidine block,colchicine treatment and serum starvation.Synchronized growth was monitored by flow cytometry.All the synchronized VSMC＇s distribution ration was （89.22±3.54） % at G0/G1 phase,（66.74 ±7.16）% at G1/S phase,（63.24 ±4.06）% at S phase and （51.64 ± 11.18）% at G2/M phase and there was statistically significant difference compared with other phase（P 〈0.01）.There was no significant effect of Cyclin A on ORC1 mRNA expression at a quiescent stage of VSMC.At G2/M phase peaked ORC1 was （52.133 3 ± 2.122 1）%,at G1/S phase was（10.916 7 ± 0.531 1）%,at S phase was （7.656 7 ± 0.412 4）%,and there was statistically significant difference （P 〈0.01）.At G2/M phase ORC1 downed to the lowest point was （1.276 7 ± 0.161 7） %,at G1/S phase was （13.371 0 ± 1.057 3）%,at S phase was （3.043 3 ± 0.538 0）%,and there was statistically significant difference （P 〈 0.01）,suggesting that Cyclin A might prevent ORC1 binding the chromatin of VSMCs.Conclusion Cyclin A may be an important regulative factor at the initiation of ORC1 in VSMCs.