CD47抗体对异丙肾上腺素诱导的大鼠心肌细胞损伤的保护作用

The protective effect of CD47 antibody on myocardial cell injury induced by Isoproterenol in rats

目的观察CD47抗体对异丙肾上腺素(ISO)诱导大鼠原代心肌细胞损伤的保护作用。方法分离培养40只1~3日龄的SD大鼠心肌细胞,使用ISO诱导原代大鼠心肌细胞损伤模型。采用随机数字表法将大鼠心肌细胞分为正常对照组(Control组)、模型组(ISO组)、治疗组(Anti-CD47+ISO组)。Control组,心肌细胞常规培养基培养;ISO组,常规培养基培养,加入ISO(10^(-5) mol/L)继续培养48 h;Anti-CD47+ISO组,常规培养基加入CD47抗体(7μg/m L)培养12 h后,加入ISO(10^(-5)mol/L)继续培养48 h。测定培养细胞上清液乳酸脱氢酶(LDH)、肌酸激酶(CK)、超氧化物歧化酶(SOD)、丙二醛(MDA)和一氧化氮(NO)含量;使用Annexin-V与PI双染法及流式细胞仪检测心肌细胞凋亡率;超氧化物阴离子荧光探针(DHE)染色检测心肌细胞活性氧(ROS)水平;Western blot检测心肌细胞CD47、Bcl-2和Bax蛋白的表达。结果与Control组比较,ISO组心肌细胞的凋亡率明显升高(P<0.01);与ISO组比较,Anti-CD47+ISO组可显著降低细胞凋亡率(P<0.01)。与Control组比较,ISO组LDH、CK、MDA和ROS水平显著升高(P<0.01),SOD和NO水平显著降低(P<0.01);与ISO组比较,Anti-CD47+ISO组SOD和NO水平显著提高(P<0.01),LDH、CK、MDA以及ROS水平显著降低(P<0.01)。Western blot结果显示,与Control组比较,ISO组CD47和Bax表达显著升高(P<0.05、P<0.01),Bcl-2表达显著降低(P<0.01);相比于ISO组,Anti-CD47+ISO组CD47和Bax表达明显降低(P<0.01),Bcl-2表达明显升高(P<0.05)。结论 CD47抗体可能通过抑制ROS的产生、调节Bcl-2/Bax蛋白表达比例来抑制心肌凋亡,从而对ISO诱导的大鼠原代心肌细胞损伤产生保护作用。

Objective To investigate the protective effect of CD47 antibody on Isoproterenol （ISO） induced primary eardiomyocytes injury in rats. Methods The myocardial cells were isolated from 40 SD rats （1-3 days old）. The model of primary myocardial cells injury was induced by ISO. The myocardial cells of rats were divided into normal control group （control group）, model group （ISO group） and treatment group （Anti-CD47＋ISO group） by random number table. In control group, myocardial cells were cultured in the conventional medium; in ISO group, the conventional culture medium was added with ISO （10^-5mol/L） for 48 h; in Anti-CD47＋ISO group, CD47 antibody （7 μg/mL） was added to the conventional culture medium for 12 h, followed by ISO （10 5 tool/L） for another 48 h. Cultured supernatants were collected to measure lactic dehydrogenase （LDH）, creatine kinase （CK）, superoxide dismutase （SOD）, malonadehyde （MDA）, nitric oxide （NO）; the myocardial apoptosis rate was detected using Annexin-V and PI double staining and flow cytometry; dihydroethidium （DHE） staining was used to detecte reactive oxygen species （ROS） in cardiomyocytes; the expression of CD47, Bcl-2 and Bax was detected by Western blot. Results Compared with the control group, the myocardial cells apoptosis rate in ISO group was significantly increased （P 〈 0.01）; compared with the ISO group, the cardiacmyocyte apoptosis rate in Anti-CI）47＋ISO group was significantly decreased （P 〈 0.01）. Compared with the control group, the levels of LDH, CK, MDA and ROS in ISO group were significantly increased （P 〈 0.01）, the levels of SOD and NO were significantly de-creased （P 〈 0.01）; compared with the ISO group, the levels of SOD and NO in Anti-CD47＋ISO group were significant- ly increased （P 〈 0.01） and the levels of LDH, CK, MDA and ROS were significantly decreased （P 〈 0.01）. The results of Western blot showed that compared with the control group, the expression of CD47 and Bax in ISO group was in- creased significantly （P 〈 0.05, P 〈 0.01）, the expression of Bcl-2 in ISO group was decreased significantly （P 〈 0.01）; compared with ISO group, the expression levels of CD47 and Bax in Anti-CD47＋ISO group were decreased significant- ly （P 〈 0.01）; the expression level of Bcl-2 in ISO group was increased significantly （P 〈 0.05）. Conclusion CD47 an- tibody may inhibit myocardial apoptosis by inhibiting the production of ROS and regulating the expression rate of Bcl- 2/Bax protein, thus playing a protective effect on ISO-induced primary myocardial cell injury in rats.