荧光共振能量转移技术检测蛋白激酶A在MIN6细胞分泌胰岛素机制中的作用  

The role of PKA in insulin secretion of MIN6 cells measured by fluorescence resonance energy transfer biosensor technology

作　　者：安学院 丁艳洁[1] 李思源[1] 李军[2] 常子涛 AN Xue-yuan;DING Yan-jie;LI Si-yuan;LI Jun;CHANG Zi-tao(Shihezi University Scool of Medicine,Shihezi 832000,Xinjiang,China;Department of Endocrinology,the First Affiliated Hospital of the Medical College,Shihezi University,Shihezi 832000,Xinjiang,China;Department of Pathology,Hebi People's Hospital,Hebi 458000,Henan,China)

机构地区：[1]石河子大学医学院,石河子832000 [2]石河子大学第一附属医院内分泌代谢科,石河子832000 [3]鹤壁市人民医院病理科,鹤壁458000

出　　处：《医学研究生学报》2021年第1期26-29,共4页Journal of Medical Postgraduates

基　　金：新疆生产建设兵团区域创新引导计划项目(2018BB040);石河子大学成果转化与技术推广项目(CGZH201911)。

摘　　要：目的蛋白激酶A(PKA)参与小鼠胰岛β细胞株MIN6细胞分泌胰岛素的机制尚不明确。文中旨在不同浓度的葡萄糖环境下,给予不同浓度的胰升血糖素和异丙肾上腺素(ISO)后MIN6细胞胰岛素和PKA水平变化,探讨PKA在MIN6细胞中分泌胰岛素的作用和机理。方法体外培养MIN6细胞,在葡萄糖0、2.8、16.7 mmol/L时分别给予胰升血糖素0、500、1000 ng/L干预处理,用荧光共振能量转移(FRET)的生物传感器技术测PKA水平,用ELISA测胰岛素水平,并在添加ISO前后,检测并比较PKA和胰岛素的变化水平。结果葡萄糖浓度2.8、16.7 mmol/L时,500 ng/L胰升血糖素(ISO+)、1000 ng/L胰升血糖素(ISO-)PKA的表达水平较500 ng/L胰升血糖素(ISO-)明显升高(P<0.05);1000 ng/L胰升血糖素(ISO+)PKA的表达水平较500 ng/L胰升血糖素(ISO+)、1000 ng/L(ISO-)明显升高(P<0.05)。0 mmol/L葡萄糖时,1000 ng/L胰升血糖素的胰岛素增量较0 ng/L胰升血糖素明显升高(P<0.05)。2.8、16.7 mmol/L葡萄糖时,500 ng/L、1000 ng/L胰升血糖素的胰岛素增量较0 ng/L胰升血糖素明显升高(P<0.05)。未添加ISO时,细胞上清液的胰岛素含量与PKA呈正相关(r=0.810,P<0.05);添加ISO后,细胞上清液cAMP与胰岛素含量亦呈正相关(r=0.791,P<0.05)。结论基于FRET的生物传感器技术可在活细胞上实时动态检测PKA的变化,胰升血糖素以浓度递增的形式通过增加MIN6细胞内PKA的浓度来促进胰岛素分泌,且这种作用具有一定的葡萄糖依赖性。Objective The mechanism of protein kinase A(PKA)in the secretion of insulin in MIN6 cells is unclear.To investigate the administration of different concentrations of glucagon(Glg)and isoprenaline(ISO),a stimulator of PKA signaling pathway,the levels of Insulin(Ins)and PKA in MIN6 cells were detected to elucidate the role and mechanism of PKA in the secretion of Ins in MIN6 cells under different glucose conditions.Methods MIN6 cells were cultured in vitro and treated with Glg 0,500,and 1000 ng/L intervention when glucose was 0,2.8,and 16.7 mmol/L.Fluorescence resonance energy transfer(FRET)biosensor technology was used to detect PKA levels.Enzyme linked immunosorbent assay(ELISA)was used to detect the level of Ins,and the changes in PKA and Ins levels were detected after adding ISO.Results The PKA level in 2.8 and 16.7 mmol/L Glu,1000 ng/L Glg iso-group and 500 ng/L Glg ISO+group were higher than that in 500 ng/L Glg iso-group(P<0.05).The PKA level in 1000 ng/L Glg ISO+group were higher than that in 1000 ng/L Glg iso-group and 500 ng/L Glg iso-group(P<0.05).The Ins level in 2.8 and 16.7 mmol/L Glu,the 1000 ng/L Glg iso-group was higher than that in 500 ng/L Glg iso-group(P<0.05).The Ins level was positively association with the level of PKA without ISO treatment(r=0.810,P<0.05).And the cAMP level was positively association with the level of Ins(r=0.791,P<0.05).Conclusion FRET-based biosensor technology can dynamically and real-timely detect the change of PKA on live cells.Glg can promote the secretion of Ins by increasing the concentration of PKA in MIN6 cells under the condition of increasing concentration,which has a certain glucose-dependent effect.

关 键 词：胰升血糖素 MIN6细胞 蛋白激酶A 胰岛素 荧光共振能量转移 

分 类 号：R44[医药卫生—诊断学]