Tang Xiao-long, Xiao Rui, Wang Yun-xiu, He Min, Xie Ting, Zhang Cheng, Liu Si-jing
Clinical Laboratory, Guangdong Provincid Hospital of Traditional Chinese Medicine, Guangzhou 510120, China.
Beijing Da Xue Xue Bao Yi Xue Ban. 2011 Jun 18;43(3):421-6.
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.
PDX1 gene and NGN3 were constructed with PAX4 genes expression adenovirus vectors, Adxsi-CMV-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.
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-NGN3/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 1 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.
NGN3 and PAX4 have a favourable role in PDX1 inducing mesenchymal stem cells into pancreatic secretory cells.
探讨神经源蛋白3（NGN3）和配对盒基因4（PAX4）在PDX1驱动间充质干细胞（MSCs）向胰腺β细胞分化过程中的作用。
构建携带PDX1基因和NGN3与PAX4基因的表达腺病毒载体，Adxsi-CMV-PDX1腺病毒感染诱导MSCs。1周后，再次用Adxsi-CMV-NGN3/CMV-PAX4腺病毒感染诱导MSCs；并检测PDX1、PAX4、NGN3、NK转录因子相关基因家族2位点2（NKX2.2）、v-maf肌腱膜纤维肉瘤癌基因同源物B（MafB）、胰岛素、胰高血糖素等胰腺分泌细胞相关分子的表达。
成功构建Adxsi-CMV-PDX1和Adxsi-CMV-NGN3/CMV-PAX4腺病毒载体。通过免疫细胞化学和间接荧光检测，在Adxsi-CMV-PDX1和Adxsi-CMV-NGN3/CMV-PAX4诱导的MSCs细胞核中稳定检测到PDX1、NGN3和PAX4因子的表达。转染5 d后，细胞形成圆形，聚集在一起，经双硫腙染色呈鲜红色。RT-PCR检测显示诱导1周后有NeuroD1和NKX2.2表达，诱导2周后有胰岛素/胰岛素原分子表达。诱导细胞可表达一些转录因子如NKX2.2和MafB，也可表达胰腺分泌相关分子如胰岛素和胰高血糖素，但不表达MafA和C肽。
NGN3和PAX4在PDX1诱导间充质干细胞向胰腺分泌细胞分化过程中具有促进作用。
