| 摘要: |
| 目的 探讨西罗莫司对体外培养的大鼠血管平滑肌细胞(VSMCs)增殖、起始识别复合物1(ORC1)表达的影响及机制。方法 细胞随机分为对照组和实验组,每组3瓶/孔,血清饥饿法培养细胞24 h使其同步化。对照组采用含10％胎牛血清的DMEM培养液培养,实验组采用含10 μmol/L西罗莫司＋10％胎牛血清的DMEM培养液培养。继续培养0～48 h,采用免疫细胞化学法检测增殖细胞核抗原(PCNA)表达,电子显微镜观察细胞超微结构,RT-PCR法检测ORC1 mRNA的表达，蛋白质印迹法检测P53、cyclin D1、cyclin A和ORC1蛋白的表达。结果 与对照组相比,实验组48 h PCNA阳性表达率降低\[(20±2.1)% vs (80±3.0)%, P＜0.05\],P53蛋白表达升高 (P＜0.05)；细胞核有固缩的迹象；实验组cyclin D1蛋白表达轻微升高(P>0.05),48 h cyclin A蛋白的表达下降(P＜0.05)。对照组ORC1 mRNA的表达在0～12 h升高,24～48 h降低,高峰期在12 h。实验组细胞ORC1 mRNA的表达始终处于高水平。与同时间对照组比较,实验组48 h ORC1 mRNA的表达升高(P＜0.05)。蛋白质印迹分析结果 与之相似。结论西罗莫司抑制VSMCs增殖,同时促进其凋亡；其作用环节可能是通过阻止细胞由G0/G1期向S期转化,诱导细胞处于静止状态；ORC1的作用环节位于西罗莫司作用点的上游,进一步支持ORC1参与细胞复制起始过程。 |
| 关键词: 西罗莫司 血管平滑肌细胞 细胞增殖 起始识别复合物1 |
| DOI：10.3724/SP.J.1008.2011.0960 |
| 投稿时间：2011-07-29修订日期：2011-08-30 |
| 基金项目:国家自然科学基金(30470727). |
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| Effect of sirolimus on cell proliferation and expression of origin recognition complex 1 in vascular smooth muscle cells of rats |
| WANG Qian1,2,SHU Mao-qin2* |
(1. Department of Cardiovasology, Changhai Hospital, Second Military Medical University, Shanghai 200433, China
2. Department of cardiology, southwest hospital, the third military medical university. Chongqing, china 400038
*Corresponding author.) |
| Abstract: |
| ObjectiveTo study the effect and mechanism of sirolimus on cell proliferation and expression of origin recognition complex 1 (ORC1) in rat vascular smooth muscle cells (VSMCs) in vitro. MethodsVSMCs were randomly divided into two groups, three bottles/well per group; synchronization of cells was achieved by serum starvation. Control group was cultured in DMEM with 10% FBS, and experimental group was cultured in DMEM with 10 μmol/L sirolimus plus 10% FBS. Expression of proliferating cell nuclear antigen (PCNA) protein was analyzed by immunocytochemistry, VSMC ultramicrostructure was observed under electron microscope, and expressions of P53,cyclin D1, cyclin A, and ORC1 mRNA and protein were displayed by RT-PCR and Western blotting analysis during 0-48 h of culture. ResultsCompared with control group, the positive rate of PCNA protein in experimental group was significantly decreased after 48 h culture (\[20±2.1\]% vs \[80±3.0\]%, P＜0.05), P53 protein expression was significantly increased (P＜0.05), with evidence of pyknosis. The expression of cyclin D1 was slightly increased in the experimental group (P>0.05), and the expression of cyclin A was significantly decreased after 48 h culture(P<0.05). ORC1 mRNA expression in the control group was increased during 0-12 h and decreased during 24-48 h, with the expression peaked at 12 h. In the experimental group ORC1 mRNA expression was kept at a high level, with the expression at 48 h being significantly higher than that in the control group (P<0.05); similar results were also found for ORC1 protein expression. ConclusionSirolimus can inhibit the proliferation of VSMCs and promote their apoptosis. The inhibition of VSMCs proliferation may be through preventing cell transformation of G0/G1 phase to S phase and keeping cells at resting phase. The effect of ORC1 in cell cycle occurs at the upstream of sirolimus, which further supports that ORC1 participates in the initiation process of DNA replication. |
| Key words: sirolimus vascular smooth muscle cells cell proliferation origin recognition complex 1 |
