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本研究探讨胰十二指肠同源框因子-1(Pdx1)基因修饰的人脐带间充质干细胞(MSCs)体外分化为胰岛β样细胞。采用携带Pdx1基因的重组腺病毒感染MSCs 7d,联合细胞因子分阶段诱导分化。RT-PCR及Westernblot、免疫细胞化学法检测诱导后细胞胰岛素相关基因表达变化;化学发光法检测诱导后细胞培养液上清中胰岛素和C肽的分泌水平。用流式细胞术检测胰岛素阳性细胞百分率。结果显示:诱导转入Pdx1基因的人脐带MSCs后,梭形细胞聚集形成胰岛样细胞团,双硫腙染色胞浆呈亮红色;诱导后的细胞表达Pdx1、胰岛素、C肽和葡萄糖转运体2基因;诱导细胞分泌胰岛素和C肽,并且在高糖作用后,胰岛素和C肽分泌增加;诱导后胰岛素阳性细胞百分率为(11.61±4.83)%。结果表明,Pdx1修饰人脐带MSCs在体外能够诱导分化为胰岛β样细胞。
This study was designed to investigate the in vitro differentiation of human umbilical cord mesenchymal stem cells (MSCs) with pancreaticoduodenal homeobox factor-1 (Pdx1) gene into islet β-like cells. MSCs were infected with the recombinant adenovirus carrying Pdx1 gene for 7 days. The combined cytokines were induced to differentiate in stages. The expression of insulin-related genes was detected by RT-PCR, Western blot and immunocytochemistry. The levels of insulin and C-peptide in the supernatants were detected by chemiluminescence. Flow cytometry was used to detect the percentage of insulin-positive cells. The results showed that after inducing Pdx1 gene into human umbilical cord MSCs, the spindle cells aggregated to form islet-like cell clusters with dithizone staining of the cytoplasm bright red. The induced cells expressed Pdx1, insulin, C-peptide and glucose transporter 2 The cells were induced to secrete insulin and C-peptide, and the secretion of insulin and C-peptide increased after high glucose treatment. The percentage of insulin-positive cells after induction was (11.61 ± 4.83)%. The results showed that Pdx1-modified human umbilical cord MSCs can differentiate into pancreatic β-like cells in vitro.