显性负效应在胰岛素基因突变所致青少年型糖尿病中的作用  

Role of dominant-negative effects in mutant INS-gene induced diabetes of youth

作　　者：孙楠[1] 赵红丽[1] 罗蕴之[1] 崔景秋[2] 王新玲[1] Sun Nan;Zhao Hongli;Luo Yunzhi;Cui Jingqiu;Wang Xinling(Department of Endocrinology,People＇s Hospital of Xinjiang Uygur Autonomous Region,Urumqi 830001,China)

机构地区：[1]新疆维吾尔自治区人民医院内分泌科,乌鲁木齐830001 [2]天津医科大学总医院内分泌代谢病科

出　　处：《中华糖尿病杂志》2018年第8期521-525,共5页CHINESE JOURNAL OF DIABETES MELLITUS

基　　金：新疆维吾尔自治区自然科学基金（2015211C196）

摘　　要：目的探讨显性负效应（指由于基因突变导致突变型蛋白自身无生理功能且影响另一正常蛋白发挥生理功能的一种干涉现象）在不同类型胰岛素基因突变所致青少年型糖尿病（MIDY）发病机制中的作用。方法观察3个与MIDY相关的突变型前胰岛素原C（A7）Y、G（BS）S、R（SP6）H对共存的野生型前胰岛素原分泌的影响，并引人一个含有MIDY相关突变但不能形成二硫键的突变型前胰岛素原．DelCys，共分为7组：分别用含有突变型前胰岛素原cDNA的重组质粒和含有人野生型前胰岛素原cDNA的重组质粒共转染293T细胞，设为C（A7）Y组、G（B8）S组、R（SP6）H组和DelCys组，将含有人野生型前胰岛素原cDNA的重组质粒与含有小鼠野生型前胰岛素原cDNA的重组质粒共转染作为正常对照组，将含有人野生型前胰岛素原cDNA的重组质粒与pCMS-EGFP质粒共转染作为阳性对照组，用含有小鼠野生型前胰岛素原cDNA的重组质粒转染293T细胞作为阴性对照组。转染48h后收集细胞和培养液，采用人特异性胰岛素原放免试剂盒检测细胞内和培养液中人胰岛素原水平。统计学分析采用单因素方差分析及t检验。结果比较细胞内人胰岛素原水平，各组间差异无统计学意义（F=0．58，P〉0．05）。比较细胞培养液中人胰岛素原水平，各组问差异有统计学意义（F=297．57，P〈0．01）。与正常对照组相比，C（AT）Y组和G（BS）S组细胞培养液中人胰岛素原水平明显降低[分别为（135．84±1．89）比（29．28±6．85）、（33．62±10．52）pmol／L，t=22．58、21．66，均P〈0．01]，R（SP6）H组和DelCys组与正常对照组相比差异无统计学意义（t=0．00、0．39，均P〉0．05）。结论与MIDY相关的突变型胰岛素原C（A7）Y和G（B8）S会对共存的野生型胰岛素原产生显性负效应（表现为抑制其胰岛素原分泌），而R（SP6）H突变不产生显性Objective Dominant-negative effect refers to an interference pattern that the mutated protein does not only perform physiological function, but also inhibits activity of co-existing normal protein. The aim of this study is to investigate the role of dominant-negative effects in mutant INS-gene induced diabetes of youth （MIDY） caused by different types of insulin gene mutations. Methods The inhibition of co-expressed wild-type proinsulin secretion by three MIDY mutants was observed and a proinsulin-DelCys was introduced which carried MIDY mutations but couldn＇t form disulfide bond. There were 7 groups： 293T cells were co-transfeeted with wild-type human preproinsulin and mutant preproinsalins [as C（AT）Y group, G （B8）S group, R（SP6）H group and DelCys group]. Wild-type mouse preproinsulin and wild-type human preproinsulin were co-transfected as normal control group. Wild-type human preproinsulin and pCMS-EGFP plasmid were co-transfected as positive control group and wild-type mouse preproinsulin transfected 293T cells as negative control group. After 48 h, the media and cell were collected. Secreted human proinsulin was measured with human-specific proinsulin radioimmunoassay. Data were analyzed with one-way ANOVA and t test. Results There was no statistically significant difference between the groups in the comparison of intracellular proinsulin levels （F=0.58, P〉0.05）. The differences of human proinsulin in cell culture medium among groups were statistically significant （F=297.57, P〈0.01）. Compared with normal control group, mutants C（A7）Y and G（B8）S blocked the secretion of co-expressed human proinsulin [（135.84 ± 1.89） vs （29.28±6.85）, （33.62± 10.52） pmol/L, respectively, t=22.58, 21.66, all P〈0.01]. There was no statistically significant difference between the normal control group and mutants R（SP6）H and DelCys groups （t=0.00, 0.39, all P〉0.05）. Conclusion MIDY mutants C（A7）Y and G（BS）S could induce the dominant-negative effects on co-ex

关 键 词：糖尿病 Β细胞功能衰竭 显性负效应 胰岛素原错误折叠 

分 类 号：R-332[医药卫生]