大鼠脂肪干细胞体外诱导分化为胰岛样细胞

Rat adipose derived stem cells differentiate into islet-like cells in vitro

目的：探索大鼠脂肪干细胞（ADSCs）向胰岛样细胞诱导分化的诱导方案。方法：取SD大鼠腹股沟区脂肪组织，酶消化法分离培养ADSCs，观察细胞形态，流式细胞仪检测表面抗原；应用4种方案［NA、 NG、 AG、 NAG， N：尼克酰胺；A：活化素A； G：胰高血糖素样肽-1 （GLP-1）］进行诱导，观察形态变化，ELISA法检测胰岛素及C肽分泌量，行双硫腙染色，RT-PCR检测胰岛β细胞相关基因。结果：ADSCs长梭形，CD44高表达，CD49d低表达，CD31、 CD106阴性；诱导7d细胞突起变短呈鹅卵石样，14d AG、 NAG组细胞近圆形，折光性增强，21d NAG组出现明显的胰岛样细胞团。ELISA显示21d NAG组胰岛素及C肽释放量明显高于其他组。双硫腙染色21d NAG组细胞团呈砖红色阳性表达。RT-PCR 14d仅AG和NAG组检测到胰十二指肠同源盒-1基因（PDX1）， 21d AG和NAG组均可检测到葡萄糖转运蛋白2（GLUT2）、 insulin 2、 insulin 1、 PDX1，但表达水平有差异，NAG组接近正常鼠胰岛β细胞，明显高于AG组。结论：ADSCs具有干细胞特性，在NAG诱导条件下可分化为有功能的胰岛样细胞。

Objective： To explore reliable methods to induce rat adipose derived stem cells （ADSCs） into islet like cells. Methods： Rat AI2kqCs dissociated from inguinal adipose tissue were cultured, and identified by morphological features and specific surface antigens by using flow eytometry. Three different reagents, including nicotinamide （N）, activin （A） and glucagon like peptide 1 （G）, were used to induce the ADSCs towards islet-like cells with four different combinations： NA, NG, AG, and NAG. Besides of morphological change, the secretion of insulin and C-peptide was detected by ELISA assay, and zinc-ion was observed by di thizone staining. Additionally, the expressions of genes related β cell were analyzed using RT-PCR. Results： The rat ADSCs were long spindle in shape, just like fibroblast, and positive against CD44, weak positive to CD49d, and negative to CD31 and CD106. After being induced by AG and NAG for 14 days, the ceils became to round and their refraction was enhanced. One week later, the cell incubated with NAG were found to be as islet like cell clusters with positive to dithizone staining. And, the detectable insulin and C peptide of these ceils were statistically higher than those in other treated cells. The expression of pancreatic and duodenal homeobox 1 （PDX1） gene was detected only in AG and NAG treated cells after induction being maintained for 14 days. The expressions of glucose transporter 2 （GLUT 2）, insulin 2, insulin 1 and PDX1 were detectable in AG and NAG group on day 21. However, expression levels of these genes in AG treated cells were significantly lower than those in cells induced by NAG, which were similar to those in normal islet of rats. Conclusion： The rat ADSCs possesses stem cell properties, and can be differentiated into functional islet-like cells with the presence of NAG.