Changes of insulin and nestin expression during the differentiation of human umbilical cord mesenchymal stem cells into islet-like cells

doi:10.3969/j.issn.2095-4344.2016.50.008

Abstract

Abstract:

BACKGROUND: Domestic and international studies have confirmed that human umbilical cord mesenchymal stem cells could be induced to differentiate into islet-like cells, but little is reported about the changes of insulin and nestin expressions during the differentiation phase.
OBJECTIVE: To observe the changes of insulin and nestin expressions during the differentiation of human umbilical cord mesenchymal stem cells into islet-like cells.
METHODS: Human umbilical cord mesenchymal stem cells were cultured using UltraCULTURE medium in vitro. Stem cells were cultured for three generations to observe cell morphological changes under an inverted microscope, to test immunophenotype by flow cytometry, and to identify the capacity of osteogenesis and adipogenic differentiation. Induction protocol was divided into two stages. In stage 1, stem cells were induced for 14 days in the UltraCULTURE medium with 4 nmol/L activin A, 25 μg/L epidermal growth factor, 100 μg/L β-nerve growth factor, 10 mmol/L nicotinamide. In stage 2, the cells were cultured in the UltraCULTURE medium with 1% insulin-transferin-selenium, 10 mmol/L nicotinamide, 10 μg/L basic fibroblast growth factor for an additional 14 days. The expressions of nestin and insulin in those differentiated cells were tested by flow cytometry, and zinc ion expression in the islet-like cell clusters was identified by dithizone staining.
RESULTS AND CONCLUSION: During the differentiation process, the insulin level was increased gradually in the induction group and reached a higher level on day 28, but the insulin expression showed negative in the control group. In addition, on day 14 of induced differentiation, the nestin expression reached the peak and then gradually reduced along with the prolonged inductive time. On day 28 of induction, islet-like cell clusters formed and were positive for dithizone staining. In this experiment, the umbilical cord mesenchymal stem cells were successfully induced and differentiated into islet-like cells, accompanied with the variation of insulin and nestin expression.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Umbilical Cord, Mesenchymal Stem Cells, Insulin-Secreting Cells, Insulin, Tissue Engineering

CLC Number:

R394.2

Shen Yi, Wang Yi-zhong, Shi Han, Shan Xia, Wang Lin-lin, Cui Xiao-lan. Changes of insulin and nestin expression during the differentiation of human umbilical cord mesenchymal stem cells into islet-like cells[J]. Chinese Journal of Tissue Engineering Research, 2016, 20(50): 7500-7506.

Figures/Tables 2

2.1 诱导前人脐带间充质干细胞的形态学特征如图1所示，脐带华通胶组织块在T 75 cm培养瓶中培养至 14 d，组织块周围爬出短小细长梭形的细胞，将组织块刮除后，待细胞融合80%传代培养。第3代脐带间充质干细胞形态呈长梭形，排列整齐，均匀贴壁成长，细胞增殖活力良好，符合进一步诱导实验要求。 2.2 人脐带间充质干细胞流式细胞仪鉴定结果如图2所示，流式细胞仪检测脐带间充质干细胞阴性表达CD14- FITC(0.15%)、CD34-PE(0.20%)、HLA-DR-APC (0.04%)，阳性表达CD44-FITC(99.86%)、CD90-PE(99.02%)、CD105-APC(99.8%)，符合干细胞表面标志[6]。 2.3 人脐带间充质干细胞成脂成骨细胞诱导分化染色鉴定如图3所示，脐带间充质干细胞加入成骨诱导液诱导9 d进行茜素红染色，在倒置显微镜下可见多个散在分布的、大小形态各异的棕红色钙化结节(图3A)。脐带间充质干细胞加入成脂诱导液诱导2周进行油红O染色，在倒置显微镜下可见细胞内出现小脂泡呈红色(图3B)。 2.4 人脐带间充质干细胞诱导分化为胰岛样细胞过程中的形态变化如图4所示，诱导分化培养第1天，可以看到细胞形态短小，呈贴壁生长，与诱导前细胞相比，未见明显差异；诱导培养第7天，细胞胞体变细长，开始呈现纤维状，漩涡形排列；诱导至14 d，可明显观察到细胞呈神经纤维样，排列整齐，均匀贴壁生长；诱导至21 d，可以明显观察到部分细胞开始聚集抱团生长；诱导至28 d，细胞由原来的纤维状或长梭形聚集为胰岛样细胞团。 2.5 人脐带间充质干细胞诱导分化为胰岛样细胞过程中巢蛋白的表达如图5所示，诱导前及诱导后第1，7，14，21，28天流式细胞仪检测巢蛋白表达，结果显示第1天巢蛋白表达为31.11%，第7天巢蛋白表达为66.78%，第14天巢蛋白表达为88.84%，第21天巢蛋白表达为72.08%，第28天巢蛋白表达为43.61%，表明细胞诱导中出现了巢蛋白阳性细胞的中间过程，符合脐带间充质干细胞分化为胰岛样细胞的诱导变化[7-8]。 2.6 人脐带间充质干细胞诱导分化为胰岛样细胞过程中胰岛素的表达如图6所示，诱导前及诱导后第1，7，14，21，28天流式细胞仪检测胰岛素表达，结果显示第1天胰岛素表达为0.81%，第7天胰岛素表达为1.72%，第14天胰岛素表达为3.07%，第21天胰岛素表达为23.58%，第28天胰岛素表达为57.02%，表明诱导生成的胰岛样细胞可表达胰岛素，并且随着诱导时间延长，表达量增高。 2.7 双硫腙染色鉴定锌离子表达结果诱导第28天细胞用双硫腙染液染色鉴定，胰岛β细胞胞浆中高浓度的锌离子是其分泌胰岛素调节血糖所必须的基本条件之一，双硫腙能特异地与锌离子螯合，形成棕色络合物[9]。如图7所示，与对照组相比，诱导组细胞双硫腙染色形成棕色络合物。"

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