蛋白激酶C抑制剂对精氨酸升压素调控心脏成纤维细胞增殖及p27蛋白表达的影响

Effects of chelerythrine on cell proliferation and p27 expression of cardiac fibroblasts modulated by arginine vasopressin

目的 观察蛋白激酶C(PKC)抑制剂白屈菜红 (chelerythrine)对精氨酸升压素 (AVP)介导的心脏成纤维细胞 (CF)增殖及p2 7蛋白表达的影响 ,探讨AVP促CF增殖的细胞内信号传导机制。方法 以培养的新生SD大鼠CF为实验模型 ,实验分 3组基础状态组 ;10 -7mol/LAVP组 ;10 -7mol/LAVP +10 -6mol/L白屈菜红组。每组 4只SD大鼠。采用胰酶消化、差速贴壁法培养CF ,四氮唑盐(MTT)比色法检测细胞增殖 ,碘化丙啶标记细胞DNA、p2 7蛋白的单抗和标记了FITC的二抗标记细胞内的p2 7蛋白 ,采用流式细胞分析仪 (FCM)技术测定细胞周期及p2 7蛋白表达的阳性率。结果 ( 1)10 -7mol/LAVP和 10 -6mol/L白屈菜红共同作用 48h ,MTT比色法测定的CF的A值 ( 0 32± 0 0 1)明显低于 10 -7mol/LAVP组 ( 0 39± 0 0 1,P <0 0 1) ;( 2 )AVP与白屈菜红共同作用组CF的S期细胞百分率 ( 4 4%± 1 7% )和细胞增殖指数 ( 2 0 9%± 1 2 % )分别明显低于AVP组 ( 15 5 %± 1 4%和31 4%± 1 5 % ) ,而G0 /G1期细胞百分率 ( 79 1%± 1 2 % )明显高于AVP组 ( 6 8 6 %± 1 5 % ) ,两组间比较差异有非常显著意义 (P <0 0 1) ;( 3)AVP与白屈菜红共同作用组CF的p2 7蛋白表达阳性率( 91 7%± 2 2 % )明显高于AVP单独作用组 ( 6 3 3%± 1 9% ) ,二?

Objective To study the effects of chelerythrine, a protein kinase C (PKC) inhibitor, on the cell proliferation and p27 expression of cardiac fibroblasts (CFs) modulated by arginine vasopressin (AVP) and to investigate the intracellular signal transduction mechanisms of AVP in CFs. Methods The cultured CFs of neonatal Sprague-Dawley rats were divided into 3 groups: AVP group (10 -7 mol/L AVP was added into the culture), chelerythrine group (10 -6 mol/L chelerythrine and 10 -7 mol/L AVP were added into the culture), and control group. MTT assay was used to evaluate the cell proliferation. The cultured cells were collected and propidium iodide was used to label the DNA so as to identify the cell cycle. Specific mouse-versus-rat p27 protein monoclonal antibody and fluorescein isothiocyanate-labeled secondary antibody were added into the cell suspension to label the p27 protein in the cells. Flow cytometry was used to determine the distribution of cell cycles and p27 expression. Results (1) The A value of CFs measured by MTT assay in AVP+ chelerythrine group was 0.32±0.01, significantly lower than that of the AVP group (0.39±0.01, P<0.01). (2) The percentage of CFs in S stage was 4.4%±1.7% in the AVP+ chelerythrine group, lower than those of the AVP group (15.5%±1.4%, P<0.01) and control group (7.5%±1.0%). The PI of CFs was 20.9%±1.2 % in the AVP+ chelerythrine group, significantly lower than that of the AVP group (31.4%±1.5 %, P<0.01). The PI of the AVP group was significantly lower than that of the control group (26.0%±1.0%, P<0.01). The percentage of CFs in G 0/G 1 stage was 79.1%±1.2% in the AVP+1 chelerythrine group, significantly higher than that in the AVP group (68.6%±1.5%, P<0.01). The percentage of CFs in G 0/G 1 stage in the AVP group was significantly lower than that in the control group (74.0%±1.0%, P<0.01) too. (3) The expression rate of p27 protein was 91.7%±2.2% in the AVP+ chelerythrine group, significantly higher than that in the AVP group (63.3%±1.9%, P<0.01). Conclusion PKC inhibitor remarkably reverses the CFs proliferation and p27 downregulation induced by AVP. It may be involved in the intracellular signal transduction pathway of AVP in CFs.