CAJ | 학술논문

目的观测胰岛素转录调控因子PDX-1、NeuroD1及MafA对小鼠骨髓间充质干细胞(mMSC)分化为胰岛素分泌细胞的作用.方法分离培养扩增mMSC,全基因合成转录因子PDX-1、NeuroD1和MafA的碱基序列,构建含目的基因的腺病毒载体并转染293A细胞包装成功后,分别或联合感染mMSC,体外培养分化后反转录聚合酶链反应(RT-PCR)检测胰岛素基因的转录,免疫荧光鉴定胰岛素蛋白表达,ELISA检测细胞上清液胰岛素的分泌情况.结果成功构建分别含目的基因的3种腺病毒载体,导入转录调控因子的mMSC启动胰岛素基因转录,体外培养分化后可见胞质内有胰岛素合成,3种转录因子联合作用的mMSC经葡萄糖刺激后培养液上清胰岛素浓度为(112.84±9.67)mU/L,而未受染mMSC细胞上清液为(1.60±0.22)mU/L,二组比较差异有统计学意义(P<0.05).结论转录因子PDX-1、NeuroD1和MafA修饰的mMSC能启动内源性胰岛素基因转录,获得显著产胰岛素功能.
목적 관측이도소전록조공인자PDX-1、NeuroD1급MafA대소서골수간충질간세포(mMSC)분화위이도소분비세포적작용.방법 분리배양확증mMSC,전기인합성전록인자PDX-1、NeuroD1화MafA적감기서렬,구건함목적 기인적선병독재체병전염293A세포포장성공후,분별혹연합감염mMSC,체외배양분화후반전록취합매련반응(RT-PCR)검측이도소기인적전록,면역형광감정이도소단백표체,ELISA검측세포상청액이도소적분비정황.결과 성공구건분별함목적 기인적3충선병독재체,도입전록조공인자적mMSC계동이도소기인전록,체외배양분화후가견포질내유이도소합성,3충전록인자연합작용적mMSC경포도당자격후배양액상청이도소농도위(112.84±9.67)mU/L,이미수염mMSC세포상청액위(1.60±0.22)mU/L,이조비교차이유통계학의의(P<0.05).결론 전록인자PDX-1、NeuroD1화MafA수식적mMSC능계동내원성이도소기인전록,획득현저산이도소공능.
Objective To evaluate the effects of insulin gene transcription regulators PDX-1, NeuroD1 and MafA on the differentiation of bone marrow mesenchymal stem cells (mMSCs) into insulin-producing cells.Methods Murine mMSCs were isolated, cultured and expanded.The base sequences of transcription factors PDX-1, NeuroD1 and MafA were obtained by total gene synthesis and the recombinant adenovirus vectors harboring target genes constructed and transfected into packaging cell line 293A. mMSCs were infected with adenovirus separately or together, and then differentiated in vitro into insulin-producing cells. Reverse transcription-polymerase chain reaction (RT-PCR) was utilized to detect insulin gene expression, immunofluorescence for identifying the presence of insulin protein and insulin enzyme-linked immunosorbent assay (ELISA) for evaluating the secretory volume of insulin.Results The differentiation extent of mMSCs into β-cell was analyzed. The β-cell-specific transcriptional regulators and insulin gene were expressed in mMSCs after transfection. Immunofluorescent analyses revealed the activated expression of insulin in the cytoplasm of differentiated cells.A significant content of insulin was released in these cells in response to a certain concentrations of glucose stimulation.The insulin content of mMSCs infected with a combination of three transcription factors was significantly higher than that of the control group [(112.84±9.67)mU/L vs (1.60±0.22)mU/L, P<0.05].Conclusion After modification by transcriptional factors PDX-1, NeuroD1 and MafA, mMSCs can secrete insulin through starting endogenous insulin gene transcription.