神经元素3与成对盒基因4促进胰腺十二指肠同源框蛋白1诱导间充质干细胞向胰腺分泌细胞分化

摘要

目的:探索神经元素3(neurogenin 3,NGN3)与成对盒基因4(paired box gene 4,PAX4)对胰腺十二指肠同源框蛋白1(pancreatic and duodenal homeobox factor 1,PDX1)驱动间充质干细胞(mesenchymal stem cells,MSCs)向胰腺β细胞分化过程中的作用.方法:构建PDX1基因及NGN3与PAX4双基因表达腺病毒,重组腺病毒Adxsi-CMV-PDX1感染诱导MSCs1周后,再行重组腺病毒Adxsi-CMV-NGN3/CMV-PAX4感染诱导MSCs;分别检测PDX1、PAX4、NGN3、NK转录因子相关的2.2(NK transcription factor related,gene family 2,locus2,NKX2.2)、v-maf muscu-loaponeurotic fibrosarcoma oncogene homolog B(MafB)、胰岛素(Insulin)、胰高血糖素(Gucagon)多种胰腺分泌细胞相关分子表达情况.结果:成功构建腺病毒Adxsi-CMV-PDX1与Adxsi-CMV-NGN3/CMV-PAX4;MSCs经Adxsi-CMV-PDX1与Adxsi-CMV-NGN3/CMV-PAX4分步诱导后,免疫细胞化学与间接荧光检测显示PDX1、NGN3与PAX4因子在细胞核内稳定表达;重组腺病毒稳定转染5 d后,细胞开始变圆并集聚成团,用双硫腙(dithizone,DTZ)染色细胞质呈亮红色;细胞经诱导1周后,用RT-PCR检测到神经源分化因子1(neurogenic differentiation 1,NruroD1)与NKX2.2表达,2周后可检测胰岛素/胰岛素原分子;间接荧光检测显示诱导后的细胞先后开始表达NKX2.2、MafB等转录因子与胰岛素/胰岛素原、胰高血糖等胰岛分泌细胞相关分子,但未能检测到v-maf musculoaponeurotic fibrosarcoma oncogene homolog A(MafA)与C肽分子表达.结论:NGN3与PAX4对PDX1诱导间充质干细胞向胰腺分泌细胞分化具有促进作用.%Objective :To explore the role of neurogenin 3 ( NGN3 ) and paired box gene 4( PAX4) in the process of PDX1-driven mesenchymal stem cells ( MSCs) to the pancreatic β-cell differentiation. Methods : PDX1 gene and NGN3 were constructed with PAX4 genes expression adenovirus vectors. AdxsiCMV-PDX1 adenovirus infection induced MSCs. One week later, re-Adxsi-CMV-NGN3/CMV-PAX4 adenovirus infection induced MSCs; and detected PDX1 , PAX4, NGN3 , NK transcription factor related, gene family 2 , locus2 ( NKX2. 2) , v-maf musculoaponeurotic fibrosarcoma oncogene homolog B( MafB) , insulin , glucagon and other pancreatic secretory cell-associated molecule expression. Results: Adxsi-CMV-PDX1 and Adxsi-CMV-NGN3/CMV-PAX4 adenovirus vectors were constructed successfully. Through immuocytochemistry and indirect fluorescence detection, the expressions of PDX1 and NGN3 and PAX4 factors were detected stably in MSCs cellular nuclei which were induced by Adxsi-CMV-PDX1 and Adxsi-CMV-NCN3/ CMV-PAX4. After transfection for 5 d , the cells formed round , gathered into a mass and showed bright red with dithizone staining. RT-PCR detection showed NruroD1 and NKX2. 2 expression after being induced for week and insulin/proinsulin molecules after being induced for 2 week. The induced cells could express some transcription factors such as NKX2. 2 and MafB , and also pancreatic-secreting related molecules such as insulin and glucagon, but not the expressions of MafA and C-peptide. Conclusion: NGN3 and PAX4 have a favourable role in PDX1 inducing mesenchymal stem cells into pancreatic secretory cells.