王锦芝;张艳美;陈一村;蔡文锋;石刚刚;蔡聪艺;

• 1:汕头大学医学院
• 2:汕头大学医学院第二附属医院

摘要：

目的研究碘化N-正丁基氟哌啶醇(F2)对缺氧复氧(H/R)所致心肌细胞损伤的作用及蛋白激酶C(PKC)活性的影响。方法用1～3d的Sprague-Dawley(SD)新生乳鼠进行心肌细胞原代培养,取生长4～5d的心肌细胞制作H/R模型。采用非放射性同位素法测定PKC活性、蛋白免疫印迹法检测PKC蛋白转位;双抗体夹心光化学测定法和酶联免疫吸附试验(ELISA)方法分别检测培养心肌细胞上清液中肌钙蛋白(cTnI)浓度和肿瘤坏死因子-α(TNF-α)分泌观察心肌细胞损伤状况。结果心肌细胞缺氧复氧时,PKC总活性明显升高,PKCα和βⅡ均发生转位。F2和cPKC特异性抑制剂G6976可减少cTnI泄漏和TNF-α的分泌,减轻心肌细胞H/R所致的损伤;PKC激动剂thymeleatoxin(TXA)拮抗F2的心肌保护作用。结论 F2对培养心肌细胞H/R损伤具有保护作用,可能与其抑制钙依赖PKCα转位有关。

关键词： 肌细胞,心脏;蛋白激酶C;缺氧;碘化N-正丁基氟哌啶醇

Abstract: Objective To observe the change of Protein Kinase C(PKC) activity,translocation of PKC isoforms-α,βⅡ in primary cultured cardiomyocytes induced by Hypoxia/ reoxygenation(H/R),which is helpful to elucidate the molecular mechanism of N-n-butyl haloperidol iodide F2 in a cardiomyocyte H/R model.Methods Neonatal rat cardiomyocytes were primarily cultured and H/R model was preparaed,PKC activity was measured by non-radioactive detection of PKC,and the translocation pattern of PKCα and βⅡisoforms was assessed by fractionated Western-blot analysis.Measurements included cardiac troponin I(cTnI) release were measured by two-site sandwich immunoassay and levels of tumor necrosis factor-α(TNF-α)were measured by sandwich enzyme-linked immunoabsorbent assay(ELISA) method to assess the degree of injury of cultured cardiomyocytes.Results In primary cultured cardiomyocytes exposed to H/R,PKC activity significantly increased after 2 h of hypoxia and 30 min of reoxygenation,PKCα and βⅡshowed significant translocation from the soluble to the particulate fraction after H/R.Treatment with the conventional PKC inhibitor G6976 and F2 reduced the levels of cTnI and TNF-α,which is blocked by the PKCα activator thymeleatoxin.Conclusion Myocardial protection of F2 depended on PKCα inhibition.

Keywords: Myocytes,cardiac;Protein kinase C;Anoxia;N-n-butyl haloperidol iodide

基金项目: 国家自然科学基金-广东联合基金(U0932005);; 广东省自然科学基金(10151503102000048);广东省自然科学基金团队项目(9351503102001)

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