构建游离脂肪酸诱导胰岛细胞凋亡模型验证球形脂联素的拮抗作用

Construction of free fatty acid-induced islet apoptosis model to verify the antagonism of globular adiponectin

目的:构建游离脂肪酸(棕榈酸)诱导胰岛细胞凋亡模型,并观察球形脂联素对胰岛细胞凋亡的拮抗作用。方法:实验于2004-15/2005-30在中山大学附属第二医院林百欣医学研究中心完成。构建游离脂肪酸诱导胰岛细胞凋亡模型及分组:采用小鼠胰岛素瘤细胞株βNIT-1,取对数生长期的细胞,按1×105个/孔的密度接种于6孔板,细胞生长至80%融合时将随机细胞分为3组,每组3孔,分别为对照组,棕榈酸组,棕榈酸+球形脂联素组。3组培养基均含5g/L的牛血清白蛋白、体积分数为0.03胎牛血清及体积分数为0.01的无水乙醇;棕榈酸组又加含有终浓度为500μmol/L棕榈酸;棕榈酸+球形脂联素组又加终浓度为500μmol/L棕榈酸和0.5mg/L脂联素。处理48h后终止实验。采用Hochest33342和脱氧核糖核酸末端转移酶介导的缺口末端标记法检测棕榈酸诱导胰岛细胞凋亡率和凋亡指数及脂联素处理后脂性凋亡的改善情况。结果:①经Hochest33342染色检测3组小鼠胰岛素瘤细胞株βNIT-1细胞凋亡指数明显不同(F=300.270,P<0.05),棕榈酸组明显高于对照组犤(12.40±1.57)%,(2.05±0.59)%犦,说明500μmol/L棕榈酸可成功构建胰岛细胞凋亡模型,经脂联素处理后凋亡指数明显降低犤(12.40±1.57)%,(3.87±0.93)%犦,说明球形脂联素组对胰岛细胞凋亡有拮抗作用。②经脱氧核糖核酸末端转移酶介导的缺口末端标记法检测小鼠胰岛素瘤细胞株βNIT-1细胞凋亡指数3组明显不同(F=191.221,P<0.05),棕榈酸组明显高于对照组犤(26.33±5.6)%,(2.32±0.78)%犦,说明500μmol/L棕榈酸可成功构建胰岛细胞凋亡模型,经脂联素处理后凋亡指数明显降低犤(2.32±0.78)%,(4.90±0.82)%,P<0.05犦说明球形脂联素组对胰岛细胞凋亡有拮抗作用。结论:①Hoescht染色法及脱氧核糖核酸末端转移酶介导的缺口末端标记法结果均表明在体积分数为0.03胎牛血清、5g/L的牛血清白蛋白的Dulbecco改良的Eagle培养液条件下加入终浓度为500μmol/L棕榈酸处理细胞48h可成功构建胰岛细胞凋亡的细胞模型。②球形脂联素可拮抗胰岛细胞的脂性凋亡,从而发挥了其调节糖脂代谢的作用。

AIM:To construct the model of free fatty acid(palmitic acid)-induced islet apoptosis,and observe the antagonism of globular adiponectin to islet apoptosis.METHODS:The experiment was carried out in the Lin Bai-xin Medical Research Center,the Second Affiliated Hospital of Sun Yat sen University between October and December 2004.Models of free fatty acid induced islet apoptosis were constructed and grouped:The insulinoma cell strain beta NIT 1 of mice was used,cells at logarithm growth period were inoculated to 6 well plate with the density of 1× 105 cells per well;When the 80% of the cells were confluent,they were randomly divided into 3 groups with 3 wells in each group: control group,palmitic acid group and palmitic acid+ globular adiponectin group.All the culture media in the 3 group contained 5 g/L bovine serum albumin,fetal bovine serum with volume fraction of 0.03,and anhydrous alcohol with volume fraction of 0.01;Palmitic acid with the terminal concentration of 500 μ mol/L was added to those in the palmitic acid group and palmitic acid+ globular adiponectin group;Besides,0.5 mg/L adiponectin was added to that in the palmitic acid+ globular adiponectin group.The experiment ended 48 hours after treatment.The rate of palmitic acid induced islet apoptosis and the apoptosis index,and the amelioration of lipoapoptosis after treatment of adiponectin were detected with Hoescht 33342 staining and terminal deoxylnucleotidyl transferase mediated dUTP nick end labeling(TUNEL).RESULTS:① Hoescht 33342 staining showed that the insulinoma cell strain beta NIT 1 apoptosis indexes were obviously different among the 3 groups(F=300.270,P< 0.05);It was markedly higher in the palmitic acid group than in the control group[(12.40± 1.57)% , (2.05± 0.59)% ],which indicated that 500 μ mol/L palmitic acid could successfully construct model of islet apoptosis,and the apoptosis index was obviously decreased after treatment of adiponectin[(12.40± 1.57)% ,(3.87± 0.93)% ],which indicated that adiponectin had the antagonistic action to the islet apoptosis index.② Terminal deoxylnucleotidyl transferase mediated dUTP nick end labeling(TUNEL) showed that the insulinoma cell strain beta NIT 1 apoptosis indexes were obviously different among the 3 groups(F=191.221,P< 0.05);It was markedly higher in the palmitic acid group than in the control group[(26.33± 5.6)% ,(2.32± 0.78)% ],which indicated that 500 μ mol/L palmitic acid could successfully construct model of islet apoptosis,and the apoptosis index was obviously decreased after treatment of adiponectin[(2.32± 0.78)% ,(4.90± 0.82)% ,P< 0.05],which indicated that adiponectin had the antagonistic action to the islet apoptosis index.CONCLUSION:① The results of both Hoescht 33342 staining and terminal deoxylnucleotidyl transferase mediated dUTP nick end labeling(TUNEL) indicate that islet apoptosis models can be successfully constructed after adding palmitic acid with the terminal concentration of 500 μ mol/L into the Dulbecco modified Eagle culture medium containing fetal bovine serum with volume fraction of 0.03 and 5 g/L bovine serum albumin for 48 hour incubation.② Adiponectin can counteract the lipoapoptosis in islet cells, and than plays a role in regulating lipid metabolism.