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胰腺小分子活性肽对H2O2诱导体外大鼠胰岛细胞凋亡的影响

来自:中国糖尿病杂志  编辑:徐艳花 王洪敏 张振|点击数:|2013-04-24

  ·糖尿病基础研究·

  胰腺小分子活性肽对H2O2诱导体外大鼠胰岛细胞凋亡的影响

  徐艳花 王洪敏 张振 杨蕾 蔡德鸿 陈光明 陈宏

  【摘要】 目的 研究胰腺小分子活性肽对H2O2诱导体外大鼠胰岛细胞凋亡的影响。 方法 采用胶原酶消化和组织培养法分离纯化大鼠胰岛细胞,检测其纯度与活性,将胰岛细胞分为空白对照组、实验组和阳性对照组,空白对照组加入普通培养基,实验组培养基中加入不同质量浓度(100,200,300,400μg/ml)胰腺小分子活性肽,阳性对照组培养基中加入100nmol/l艾塞那肽,每组细胞做5个复孔(n=5),培养5 d后每组均接受100μmmol/LH2O2处理1 h,采用Hoechst33258核染色形态学观察;AnnexinV-FITC/PI双染色流式细胞仪检测各组细胞凋亡率;荧光定量PCR法检测bax、bcl-2及caspase3 mRNA的表达;Western-bolt检测bax、bcl-2蛋白的表达。 结果 除100、200μg/ml胰腺小分子活性肽组外,300、400μg/ml胰腺小分子活性肽和阳性对照组胰岛细胞凋亡率明显小于空白对照组(P<0.05),且后三者相比较差异无统计学意义;300μg/ml与400μg/ml胰腺小分子活性肽组胰岛细胞bcl-2蛋白表达明显高于空白对照组(P<0.05),且bax 与caspase3蛋白表达显著低于空白对照组(P<0.05);300μg/ml与400μg/ml胰腺小分子活性肽组胰岛细胞bcl-2 mRNA的表达高于空白对照组(P<0.05),且bax mRNA的表达低于空白对照组(P<0.05)。 结论 胰腺小分子活性肽在浓度为300μg/ml与400μg/ml时对大鼠胰岛细胞凋亡有明显保护作用。

  【关键词】过氧化氢;胰腺小分子活性肽;胰岛细胞;凋亡

Effect of pancreatic micromolecular active peptides on the apoptosis of the rat islet cells induced by H2O2 in vitro XU Yan-hua, WANG Hong-min, ZHANG Zhen, et al. Department of Endocrinology, Zhujiang Hospital, Southern Medical University, Guangzhou 510282, China

  【Abstract】 Objective To investigate the effect of pancreatic micromolecular active peptides on the apoptosis of the rat islet cells induced by H2O2 in vitro. Methods The rat islet cells were isolated and purified by collagenase digestion and tissue culture, and then their purity and activity were detected. The isolated and purified cells were divided into the blank control group, experimental group, and positive control group. The blank control group was put into the normal culture medium. Into the culture medium of the experimental group, the pancreatic micromolecular active peptides in different concentrations (100,200,300,400μg/ml) was added. However, into the culture medium of the positive control group, the 100nmol/l exenatine was added. Every group (5 multiple holes in each, n=5) was managed with H2O2 in the concentration of 100μmmol/L for 1 hour. Morphologic observation on the islet cells stained with Hoechst33258 was carried out. Apoptosis rate of each group was tested with AnnexinV-FITC/PI staining by flow cytometry, the expressions of bax (pro-apoptosis), bcl-2 (anti-apoptosis), and caspase3 mRNA were detected by Real-Time PCR, and bax and bcl-2 proteins determined by Western Blot. Results Except the 100μg/ml and 200μg/ml groups, the apoptosis rate of islet cells in the 300μg/ml and 400μg/ml groups and the positive control group was significantly lower than that in the blank control group (P<0.05), and there existed no difference in the comparison of the latter three groups. The expression of bcl-2 protein in the 300μg/ml and 400μg/ml groups was significantly higher than that in the black control group (P<0.05), and the expression of bax and caspase3 proteins was significantly lower than that in the blank control group (P<0.05). The expression of bcl-2 mRNA in 300μg/ml and 400μg/ml groups was significantly higher and of bax mRNA lower than in the blank control group (P<0.05). Conclusion Pancreatic micromolecular active peptides function to protect obviously the rat islet cells in the concentrations of 300μg/ml and 400μg/ml.

  【Key words】 Hydrogen peroxide (H2O2); Pancreatic micromolecular active peptides; Islet cells; Apoptosis

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