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目的探讨血管紧张素受体I拮抗剂(AT1Ra)氯沙坦对体外培养的犬胰岛细胞凋亡和凋亡相关基因表达的影响。方法机械自动化分离胰岛,纯化系统获得高纯度胰岛后,分为3组进行培养。(1)单纯培养组;(2)血管紧张素Ⅱ(AngⅡ)组:培养基中加入不同浓度的AngⅡ(0.1、1.0和10.0μmol/L);(3)氯沙坦组:在加人不同浓度的AngⅡ前30min给予氯沙坦10.0μmol/L。各组胰岛培养48h后,采用透射电镜观察胰岛细胞凋亡情况;原位末端脱氧核糖核酸转移酶标记法(TUNEL)检测胰岛细胞凋亡百分率;免疫组织化学法和流式细胞术检测胰岛细胞凋亡相关基因(Bax和Bcl-2)的表达。结果单纯培养组胰岛细胞凋亡百分率为(5.70±1.18)%,AngⅡ组在AngⅡ浓度为0.1μmol/L、1.0μmol/L和10.0μmol/L时,胰岛细胞凋亡百分率分别为(9.77±1.95)%、(16.42±3.01)%和(18.22±2.31)%,氯沙坦组分别为(6.56±1.85)%、(9.25±1.58)%和(11.20±2.48)%,AngⅡ组与单纯培养组和氯沙坦组相比较,差异有统计学意义(P<0.05)。AngⅡ组Bax和Bcl-2基因表达增强,荧光指数(FI)分别为1.86±0.15和1.43±0.07,Bax/Bcl-2比值(1.30)升高;而氯沙坦组Bax表达下调至1.56±0.14,Bcl-2表达上调至1.67±0.09,Bax/Bcl-2比值(0.93)降低,两组比较,差异有统计学意义(P<0.05)。结论AngⅡ可以诱导体外培养的犬胰岛细胞凋亡;而氯沙坦对胰岛细胞具有保护作用,其可能机制是通过影响凋亡相关基因Bax和Bcl-2的表达实现的。
Objective To investigate the effects of angiotensin receptor-1 antagonist (AT1Ra) losartan on apoptosis of islet cells and expression of apoptosis related genes in canine islets. Methods The islets were isolated by mechanical automatization and the high purity islets were obtained after the purification system was divided into three groups for culture. (2) AngⅡ group: different concentrations of AngⅡ (0.1,1.0 and 10.0μmol / L) were added to the culture medium; (3) Losartan group: Losartan 10.0μmol / L 30min prior to Ang Ⅱ concentration. The islet cells in each group were cultured for 48h, the apoptosis of islet cells was observed by transmission electron microscopy; the apoptosis percentage of islet cells was detected by TUNEL; the apoptosis of islet cells was detected by immunohistochemistry and flow cytometry Expression of death related genes (Bax and Bcl-2). Results The percentage of apoptotic islets was (5.70 ± 1.18)% in cultured group and that in Ang Ⅱ group was (9.77 ± 1.95) at Ang Ⅱ concentrations of 0.1μmol / L, 1.0μmol / L and 10.0μmol / L ), (16.42 ± 3.01)% and (18.22 ± 2.31)% respectively in the losartan group and (6.56 ± 1.85)%, (9.25 ± 1.58) and Compared with losartan group, the difference was statistically significant (P <0.05). The expression of Bax and Bcl-2 in AngⅡgroup were enhanced, the fluorescence index (FI) were 1.86 ± 0.15 and 1.43 ± 0.07 respectively, and the Bax / Bcl-2 ratio (1.30) was increased in AngⅡgroup, while the Bax expression in losartan group was decreased to 1.56 ± 0.14 , The expression of Bcl-2 was up-regulated to 1.67 ± 0.09, and the ratio of Bax / Bcl-2 was decreased (0.93). There was significant difference between the two groups (P <0.05). Conclusion Ang Ⅱ can induce the apoptosis of canine islet cells cultured in vitro. Losartan has a protective effect on islet cells. The possible mechanism is that AngⅡ can affect the expression of Bax and Bcl-2.