甘精胰岛素干预游离脂肪酸诱导RIN-m细胞凋亡的变化

Effects of insulin glargine on free fatty acid induced apoptosis of RIN-m cells

目的:观察甘精胰岛素对游离脂肪酸诱导的β细胞(RIN-m细胞)凋亡是否具有保护作用,并与普通胰岛素及胰岛素样生长因子1的干预效果进行对比。方法:本实验于2004-05/2004-10在中山大学附属第二医院林百欣医学科学研究中心完成。RIN-m细胞被培养并常规培养传代。诱导凋亡接种于6孔板或者96孔板后常规培养2d,待细胞长至70%满左右时,吸去培养基,加入含10g/L血清白蛋白,10mL/L乙醇,20mL/L胎牛血清的培养基(正常对照组),或者含10g/L血清白蛋白,体积分数为0.01乙醇、体积分数为0.02胎牛血清和0.3mmol/L棕榈酸的培养基(凋亡对照组),或者含10g/L牛血清白蛋白,体积分数为0.01乙醇、体积分数为0.02胎牛血清,0.3mmol/L棕榈酸以及100nmol/L甘精胰岛素(甘精胰岛素组)或者100nmol/L普通胰岛素(普通胰岛素组)或者10nmol/L胰岛素样生长因子1组的培养基,孵育24h后,将细胞接种于6孔板,诱导细胞凋亡。采用Hoechst33342染色、流式细胞仪以及酶联免疫吸附反应方法检测各组RIN-m细胞的凋亡,吸光度值代表细胞裂解液中核小体的数量,即DNA断裂的数量。结果:0.3mmol/L棕榈酸孵育后RIN-m细胞出现核皱缩、碎裂等典型凋亡表现。①甘精胰岛素组、普通胰岛素组、胰岛素样生长因子1组亚二倍体细胞比率(32.00±3.08)%,(35.97±3.14)%,(25.25?

AIM:To investigate whether insulin glargine has the protective effect on apoptosis induced by free fatty acid(FFA),and compare the interventional effect with those of regular insulin and insulin-like growth factor-1(IGF-1) in pancreatic beta(β) cells. METHODS:The experiment was carried out in the Lin Bai-Xin Medical Scientific Research Center of the Second Affiliated Hospital of Sun Yat-sen University from May to October 2004.RIN-m cell was cultured and kept in routine passages.Cells were inoculated in 6-well or 96-well plates for 2 days,and used for determination of apoptosis at about 70%confluence.The medium was removed and replaced with that supplemented with 10 g/L bovine serum albumin(BSA),10 mL/L ethanol,20 mL/L fetal bovine serum(FBS) in absence(normal control group) or presence of 0.3 mmol/L palmitic acid(apoptosis control group) and 100 nmol/L insulin glargine(glargine group) or regular insulin(regular insulin group) or 10 nmol/L insulin-like growth factor-1(IGF-1 group).After incubation for 24 hours,the cells were inoculated to the 6-well plate for induction of apoptosis.RIN-m apoptosis in each group was detected with Hoechst33342 staining,flow cytometry and enzyme-linked immunoadsordent assay(ELISA),and the apoptic DNA fragmentation was determined quantitatively.Absorbance,which stood for the number of nucleosome in cell lysate,that was the number of DNA fragmentation,was determined at double wavelength of 405 nm against 492 nm. RESULTS:After incubation in 0.3 mmol/L Morphological analysis of nuclei of RIN-m cells incubated with 0.3 mmol/L palmitic acid,RIN-m cells showed chromosome condensation and fragmentation of nuclei.①The percentages of hypodiploid cells(SubG1) in the insulin glargine group,regular insulin group and IGF-1 group[(32.00±3.08)%,(35.97±3.14)%,(25.25±1.66)%] were obviously higher than that in the normal control group[(3.28±1.44)%](P< 0.01),but markedly lower than that in the apoptosis control group[(42.10%±4.24)%](P< 0.01).②The numbers of nucleosome in cell lysates(absorbance value) in the insulin glargine group,regular insulin group and IGF-1 group(2.372±0.21,2.758±0.20,1.704±0.16) were obviously lower than that in the normal control group(0.341±0.023)(P< 0.01),but markedly lower than that in the apoptosis control group(3.333±0.23)(P< 0.01),and it was remarkably lower in the insulin glargine group and IGF-1 group than in the regular insulin group(P< 0.05). CONCLUSION:Insulin glargine plays a role in inhibiting apoptosis induced by FFA in pancreatic βcells,which is stronger than that of regular insulin.