胰岛新生相关蛋白对胰岛β细胞增殖和胰岛素分泌功能的影响

Effects of islet neogenesis-associated protein on quantity and function of pancreatic beta-cells

目的 观察胰岛新生相关蛋白对胰岛β细胞增殖和胰岛素分泌功能的影响.方法 INS-1细胞购自上海拜力生物科技有限公司.葡萄糖刺激实验中,INS-1细胞被分为实验组（50 mg/L胰岛新生相关蛋白与INS-1共培养）和对照组,经2.8、16.7 mmol/L含葡萄糖1640培养液刺激1 h后,采用放射免疫法检测上清液中胰岛素含量.另设立24、48 h组,分别以1、10、25、50、100、250、500 mg/L胰岛新生相关蛋白、INS-1细胞共培养,24或48 h后检测吸光度值.逆转录聚合酶链反应中,INS-1细胞被分为实验组（50 mg//L胰岛新生相关蛋白与INS-1共培养）和对照组,检测PCNA、Cyclin d1、Cdk4、P27、p38MAPK、JNK mRNA表达水平.采用t检验和单因素方差分析进行数据统计.结果 2.8 mmol/L葡萄糖刺激下,实验组胰岛素释放量高于对照组[分别为（74±16）、（39±9）mU/L,t=3.96,P＜0.05];16.7 mmol/L葡萄糖刺激下,实验组胰岛素释放量亦高于对照组[分别为（78±9）、（46±10）mU/L,t=4.72,P＜0.01].随着胰岛新生相关蛋白浓度增加,INS-1细胞增殖更为明显,具有剂量依赖性,且刺激48 h的效应强于24 h.50 mg/L胰岛新生相关蛋白干预24 h后,实验组和对照组相比,PCNA、Cyclin d1、Cdk4 mRNA表达上调（t值分别为7.64、5.98、12.87,均P＜0.01）,P27、p38MAPK、JNK mRNA表达下降（t值分别为10.61、27.64、2.95,均P＜0.05）.结论 胰岛新生相关蛋白干预后,INS-1细胞胰岛素释放水平增加,胰岛细胞数量增多,这可能与其影响细胞周期调控基因的表达有关.

Objective To explore the effects of islet neogenesis-associated protein （INGAP）on proliferation and insulin secretion of pancreatic beta-cells. Methods INS-1 cells were purchased from Shanghai Bioleaf Biotech Company. INS-1 cells were co-cultured with or without 50 mg/L INGAP for 24 h. Insulin secretion was stimulated by treatment of cells with 2. 8 or 16. 7 mmol/L glucose for 1 h. The level of insulin in the supernatant was quantized using a rat insulin RIA kit. To examine the dose-response relationship between INGAP and quantity of INS-1 cells,the cells were treated with 1,10,25,50,100,250,and 500 mg/L INGAP for 24 or 48 h. 3- （4,5-dimethylthiazol-2-yl） -2,5-diphenyltetrazolium bromide （MTT） cell proliferation assay was adopted to detect the vitality of cells. The mRNA expression of PCNA,Cyclin dl,Cdk4,P27,p38MAPK,and JNK in INS-1 cells co-cultured with or without 50 mg/L INGAP for 24 h were examined by reverse transcription and polymerase chain reaction assay. The statistical significance was determined by Student＇ s t test and One-way ANOVA. Results In response to 2. 8 mmol/L glucose,the insulin secreted by the co-culture group was more than the control group （ （74 ± 16） vs （39 ±9）mU/L,t =3.96,P〈0.05）,and under the condition of 16.7 mmol/L glucose,that was （78 ±9） vs （46 ± 10）mU/L （t = 4. 72,P 〈 0. 01 ）. MTT indicated a dose-response relationship between I NGAP and quantity of INS-1 cells,and intervention for 48 h had a stronger effect than the 24 h. In contrast to the control group,the mRNA expressions of PCNA,Cyclin d1,and Cdk4 were up-regulated in INS-1 cells co-cultured with 50 mg/L INGAP （t values were 7. 64,5.98,and 12. 87; all P 〈0. 01 ）; the mRNA expressions of P27,p38MAPK,and JNK were down-regulated （ t values were 10. 61,27.64,and 2. 95; all P 〈 0. 05 ） .Conclusions INGAP increased the glucose-stimulated insulin secretion and the prolifration of INS-1 cells.The mechanism may contribute to changed expression of some genes that are related with cell cycle.