【作者】 周海燕；

【导师】 谢红旗； 刘东波；

【作者基本信息】 湖南农业大学 ， 药用植物资源工程， 2018， 硕士

【摘要】 国际糖尿病联盟数据显示,目前世界上有4.25亿糖尿病患者,预计到2045年这个数值将上升到6.29亿,其中中国患者占1.14亿。Ⅱ型糖尿病占糖尿病总数的90%左右。Ⅱ型糖尿病的两个主要致病因素是胰岛素抵抗和后期胰岛β细胞衰竭。B族维生素在治疗Ⅱ型糖尿病及其并发症都有改善,但其治疗机制尚不清楚。本文构建RIN-葡萄糖依赖性肠促胰岛素受体（GIPR）细胞模型,用于检测B族维生素对GIPR激活引起细胞中GIPR内吞,并利用细胞免疫荧光技术和western blot检测B族维生素评价胰岛β细胞自噬。研究结果如下:1.将质粒pCMV6-AC-GIPR/GFP转染至RIN-m5F细胞中,通过G418的筛选获得单克隆细胞株,该细胞通过多次传代都能稳定遗传。在荧光显微镜下可以看到绿色荧光分布均匀。用（D-Ala²）GIP作用20min时细胞内出现荧光斑点,表明GIPR激动剂类药物的筛选模型构建成功。将该模型应用于评价B族维生素,结果显示在B族维生素(1×10^-5mol/L、1×10^-6mol/L、1×10^-7mol/L)作用于细胞后,都没有出现荧光斑点激活模型,而用（D-Ala²）GIP作用于细胞在20min和40min都出现了荧光斑点。表明B族维生素没有激活GIP受体引起细胞内吞。2将B族维生素（每种设4个浓度梯度）处理后,通过细胞免疫荧光检测发现VitaminB1作用下的细胞自噬体没有增加,而在VitaminB2、VitaminB3、VitaminB5、VitaminB6、VitaminB7、VitaminB9、VitaminB12作用下细胞自噬点明显增加。用western blot检测自噬相关蛋白,发现VitaminB2、VitaminB3、VitaminB5、VitaminB6、VitaminB7、VitaminB9、VitaminB12处理后的RIN-m5F细胞,其LC3Ⅱ/LC3Ⅰ的比值上调,P62蛋白水平下降,AMPK磷酸化水平上升,mTOR磷酸化不变,说明这7种维生素都是通过AMPK途径诱导胰岛β细胞自噬。更多还原

【Abstract】 According to the international diabetes federation（IDF）,there are currently 425 million diabetics in the world,and the number is expected to rise to 629 million by 2045.Type 2diabetes accounts for about 90%of total diabetes.The two main pathogenetic factors of type2 diabetes mellitus are insulin resistance and isletβcell failure.B group vitamins has certain effect in the treatment of type 2 diabetes mellitus and its complications,but its therapeutic mechanism is not clear.The RIN-GIPR cell model was constructed in this paper.To detect the effects of B vitamins on RIN-GIPR cells,and to detect the effect of B vitamins onβ-cell autophagy using cellular immunofluorescence and western blot.The results are as follows:1.The plasmid pCMV6-AC-GIPR/GFP was transferred into RIN-m5F cells,and the monoclonal cell line was obtained by G418 screening.The cell line could be inherited stably by multiple passages.Under the fluorescence microscope,we can see that the distribution of green fluorescence is uniform.Fluorescence spots appeared in 20min treated with（D-Ala²）GIP.The results showed that the screening model of GIPR agonists was successfully constructed.The model was applied to evaluation of B vitamins,according to the results in B vitamins(1×10^-5mol/L、1×10^-6mol/L、1×10^-7mol/L)on the cell（10 min,20 min,40 min）,fluorescence spots did not activate the model.The cells treated with GIP（D-Ala²）showed fluorescent spots in both 20min and 40min.The results showed that the action site of vitamins B ware not GIP receptor and had no effect on RIN-GIPR cells.2.Group B vitamins（4 concentration gradients each）were treated for 24 h.The results of cellular immunofluorescence assay showed that the macrophages induced by VitaminB1did not increase.The cell autophagy was significantly increased in VitaminB2,VitaminB3,VitaminB5,VitaminB6,VitaminB7,VitaminB9 and VitaminB12.Using western blot detecting autophagy related proteins and found VitaminB2,VitaminB3,VitaminB5,VitaminB6,VitaminB7,VitaminB9,VitaminB12 RIN-m5F cells after processing,its LC3Ⅱ/LC3Ⅰratio increases,P62 protein levels drop,AMPK phosphorylation levels rise,mTOR phosphorylation is changeless,that seven kinds of vitamins are AMPK islet beta cells induced by autophagy.更多还原