摘要
目的探讨miR-375对人胚胎干细胞（hESC）定向分化为胰岛素分泌细胞（IPCs）的调控作用及其潜在机制。方法体外诱导hESC定向分化为IPCs，分析miR-375与其预测靶基因肝细胞核因子1β（HNF-1β）之间表达水平的动态变化关系；以miR-375过表达的慢病毒载体感染分化第二阶段细胞，实验分为空白对照组、阴性对照（GFP）组及miR-375过表达组；应用实时定量PCR和（或）Westernblotting法检测各组分化细胞中miR-375、HNF-1β、胰岛细胞分化或功能相关的关键基因的表达。两组间比较用t检验，多组间比较用单因素方差分析和Q检验。结果hESC分化五个阶段中miR-375表达水平呈先高后低的变化趋势，相对表达量分别为100％、（472．25±33．53）％、（768．00±25．65）％、（54．25±5．74）％、（30．75±5．70）％（F=1137．57，P〈O．001）。HNF．1B表达水平在分化第三阶段开始明显上升，在分化第四阶段达到高峰【分别为（279．50±21．30）％、（645．00±64．55）％，F=224．86，P〈0．0011，变化趋势与1／miR-375的变化趋势相似。慢病毒感染后，miR-375过表达组的HNF-1β mRNA表达水平与GFP组、空白对照组无显著差异（F=1．467，P〉0．05），而miR．375过表达组的HNF-1β蛋白表达水平则下降至GFP组的（38．75±9．22）％（F=60．69，P〈0．001）。与GFP组相比，miR-375过表达组分化第四阶段细胞的胰十二指肠同源盒因子1（Pdx-1）表达水平显著升高，分化第五阶段细胞（IPCs）的Nkx6．1、配对盒因子4（Pax-4）及胰岛素的表达水平则显著降低（F=105．19～484．05，均P〈0．001）。结论miR-375对hESC向IPCs定向分化具有重要的调控作用，该作用可能是通过影响转录因子HNF-1β表达而实现的。
Objective To investigate the effect and underlying mechanism of miR-375 on regulating differentiation of human embryonic stem （hES） ceils into insulin producing cells （IPCs）. Methods hES cells were induced into IPCs in vitro. The dynamic expression of miR-375 and hepatocyte nuclear factor 1β （HNF-1β） the predicted target gene, during the differentiation was analyzed. The stage 2- cells during the differentiation infected with miR-375-overexpression lentiviral vector were divided into 3 groups： control group, GFP control group and miR-375-overexpression group. The expressions of miR-375, HNF-1β and the specific markers related to pancreatic islet differentiation or function were determined by reahime PCR and/or Western blotting. Independent sample t test was used to analyze the difference between two group. ANOVA and Q test were used to analyze the difference among multiple groups. Results miR- 375 expression in the stage 1- to 5-cells were 100%, （472.25±33.53）%, （768.00±25.65）%, （54.25±5.74）% and （30.75±5.70）%, respectively （F= 1β 7.57, P〈0.001）. The expression of HNF-1β increased from stage 3 （（279.50±21.30）%）, and peaked at stage 4 （（645.00±64.55）%,F=224.86, P〈0.001）. The dynamic level of HNF-1β was similar as that of 1/miR-375. After the lentivirus infection, the mRNA level of HNF-1β in miR- 375-overexpression group was not statistically different from that of both the GFP group and control group （F=1.467, P〉0.28）, whereas the protein level of HNF-1β decreased to （38.75±9.22）% of the GFP group in the miR-375 overexpression group （F=60.69,P〈0.001）. miR-375 overexpression upregulated the expression of pancreatic and duodenal homeobox 1 （Pdx-1） in stage 4-cells （F=412.15, P〈0.001）, and downregulated the expression of the pancreatic islet specific markers, e.g. Nkx6.1, paired box 4 （Pax-4） and insulin in the stage 5-cells （F=105.19-484.05,all P〈0.001） when compared with the GFP group. Conclusion miR-375 plays a modulatory role in the IPC differentiation from hES ceils. That may be mediated via regulating the expression of the transcription factor HNF-1β.
出处
《中华糖尿病杂志》
CAS
CSCD
2015年第12期730-734,共5页
CHINESE JOURNAL OF DIABETES MELLITUS
基金
国家自然科学基金资助项目（81400767、81400798、81270858、81401142、81570692）
教育部博士点新教师基金资助项目（20120001120076、20120001120069、20120001120063）
