胰十二指肠同源框因子-1联合细胞因子诱导人脐带间充质干细胞分化为胰岛β样细胞的研究

PDX1 gene and cytokines induce human umbilical cord mesenchymal stem cells to differentiate into islet β-like cells in vitro

目的探讨人脐带间充质干细胞（mesenchymal stem cells，MSCs）体外诱导分化为胰岛β样细胞的办法。方法用含胰十二指肠同源框因子-1基因（pancreatic and duodenal homeobox factor 1，PDX1）重组腺病毒（Adxsi—CMV—PDX1）感染人脐带MSCs，再联合多种细胞因子进行诱导分化。应用RT—PCR、免疫荧光染色法检测诱导后细胞的PDX1、胰岛素（insulin）、神经元素3（ngn3）、葡萄糖转运体2（glut2）、NK转录因子6．1（NKX6．1）mRNA的表达；化学发光法检测诱导后细胞培养上清中胰岛素和C肽的分泌精；流式细胞仪检测胰岛素阳性细胞率。结果Adxsi—CMV—PDX1感染脐带MSCs并联合细胞因子诱导后，细胞从短梭形变成长梭形，并聚集形成胰岛样细胞团，经双硫腙染成亮红色。诱导17d后的细胞表达PDX1、ngn3、NKX6．1、insulin、glut-2的mRNA；细胞胞质内有胰岛素和C肽；细胞培养上清中胰岛素和C肽含量分别为（473．1±51．5）mU／L和（1．61±0．41）ng／ml，用25mmol／L高糖刺激1h后，胰岛素和C肽分泌量高达（964．4±68．1）mU／L和（3．72±1．52）ng／ml；胰岛素阳性细胞率达（11．6±4．8）％。结论PDX1转入脐带MSCs后，联合细胞因子在体外能够诱导其分化为胰岛β样细胞，胰岛素和C从分泌水平较高，且对高糖刺激有反应，但还不是完全成熟的β细胞。

Objective To explore the method how PDX1 gene modified mesenchymal stem cells （MSCs） from human umbilical cord can be differentiated into islet β-like cells in vitro. Methods Recombined adenovirus vectors inserted with PDX1 （Adxsi-CMV-PDX1） was transfected into MSCs and multiple cytokines was combined to induce differentiation. The expressions of PDX1, insulin, ngn3, glut2, NKX6. 1 were examined by RT-PCR and immunofluorescence staining. The levels of insulin and C peptide secretion were examined by chemiluminescence immunoassay. Flow cytometry was used to determine the positive rate of insulin cells. Results After Adxsi-CMV-PDX1 transfection and cytokines induction, MSCs were transformed from short spindle shape to long spindle shape and aggregated into islet-like cell clusters. Dithizone staining of these cells showed bright red color. PDX1 , ngn3, NKX6. 1 , insulin, glut 2 mRNA were expressed in cells 17d after induction. Insulin and C peptide were expressed in cytoplasm. The levels of insulin and C peptide in cell culture supernatant were （473. 1 ± 51.5 ）mU/L and （1.61 ± 0.41 ）ng/ml; the levels of insulin and C peptide secretion were （964.4 ± 68.1 ） mU/L, （3.72 ± 1.52） ng/mL, respectively, with 2 5 mmol/L glucose stimulation for one hour. Insulin （ ＋ ） cells rate （ 11.6 ± 4.8） %. Conclusions Adxsi-CMV-PDXI combined with cytokines can induce MSCs from human umbilical cord to differentiate into islet β-like cells. They can secret insulin and C peptide, and have the sensitivity to the stimulation of glucose.