荧光分析活性氧在血管紧张素Ⅱ诱导心肌成纤维细胞增殖中的作用

Role of Reactive Oxygen Species by Fluorescence Analysis in Proliferation of Myofibroblasts Induced by Angiotensin Ⅱ

应用荧光分析方法探讨活性氧(Reactive oxygen species,ROS)在血管紧张素Ⅱ(AngiotensinⅡ,AngⅡ)诱导心肌成纤维细胞(Myofibroblasts,myoFbs)增殖中的作用。实验中采用胰酶消化法分离、培养原代新生大乳鼠myoFbs,10%胎牛血清培养,采用2~3代myoFbs,并随机分为正常对照组、AngⅡ处理组、N-乙酰半胱氨酸(N-Acetyl-L-cysteine,NAC)干预组,培养12、24和36 h后,分别采用3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐(3-(4,5)-Dimethylthiahiazo(-z-yl)-3,5-diphcnyt-etrazoliumromide,MTT)法测定myoFbs的增殖率;硝酸酶还原法检测myoFbs产生羟自由基(·OH)的量;利用荧光探针DCFH-DA分析检测myoFbs中ROS的水平;免疫细胞化学染色法及荧光共聚焦测定p-PKCα的表达及膜转位。结果显示一定浓度的AngⅡ作用myoFbs 24 h时myoFbs增殖率明显增加(η值(%)为117.05);并诱导myoFbs产生大量ROS,其中myoFbs增殖率越高,·OH的含量(94.53±1.68)越高;免疫细胞化学染色及荧光共聚焦可见,AngⅡ能明显促进pPKCα的表达及膜转位(与对照组比较p<0.01或p<0.001);NAC(10^(-4)mol/L)可抑制AngⅡ引起myoFbs的增殖(OD值为0.357±0.13),减少myoFbs内ROS及·OH的量(75.57±1.48),抑制p-PKCα的表达及膜转位。

Fluorescence analysis was performed to explore the role of reactive oxygen species（ROS） in the proliferation of myofibroblasts（myoFbs） induced by angiotensin Ⅱ（Ang Ⅱ）.Primary cultures of neonatal rat myoFbs were obtained by enzymatic dissociation of Wistar rat neonates,and myoFbs were cultured under 10%fetal bovine serum.MyoFbs of 2-3 generation cultured in vitro were used in experiments and divided into three groups which were treated by Ang Ⅱ,Ang Ⅱ ＋N-acetyl-L-cysteine（NAC）,and normal culture medium,respectively.MyoFbs were cultured for 12,24 and 36 h.In the experiment,the proliferation rate of myoFbs induced by Ang Ⅱ under different concentrations of 3-（4,5）-dimethylthiazo（-z-y1）-3,5-diphenyletrazoliumromide（MTT） was detected,the ROS levels of myoFbs were detected by fluorescent probes in 2＇,7 ＇-dichlorofluorescein fluorescence Huang Shuang acetate（DCFH-DA）,and the contents and levels of oxygen free radicals（·OH） in three groups were detected by spectrophotometry,immunocytochemical staining and fluorescence confocal.Immunocytochemical staining and fluorescence confocal analysis was used to measure membrane translocation and expression of p-protein kinase Cα（p-PKCα）.The results showed that myoFbs incubated with Ang Ⅱ（10^（-8）- 10^（-6） mol/L） for 24 h increased the proliferation rate（the value of η（%） was170.15） and ROS,especially the content of · OH（94.53 ± 1.68） reached the highest level.The immunocytochemistry,confocal fluorescence staining and image analysis result showed that Ang Ⅱ could increase the translocation and expression of p-PKCα in membrane（p 0.001 vs control group）;NAC could inhibit the proliferation of myoFbs induced by Ang Ⅱ（the value of η（%） was 117.05）,decrease the content of ROS and the levels of · OH（75.57 ＋ 1.48）,and inhibit the translocation and expression of p-PLCα in membrane.