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单纯生物学制剂诱导人脐带间充质干细胞向胰岛素分泌细胞快速分化

王跃春, 张洹, 段阿林, 林伟霞, 郑乔丹, 徐文璐

暨南大学医学院 生理学教研室； 血液病研究所， 广州 510632

摘要

为探讨用单纯生物学制剂诱导人脐带间充质干细胞(mesenchymal stem cells derived from human umbilical cord, hUCMSCs)向胰岛素分泌细胞分化的可行性，本研究用胶原酶Ⅱ、胰蛋白酶次序消化及两步离心法从人胎儿完整脐带中分离、纯化出hUC-MSCs；用表皮生长因子、碱性成纤维生长因子、银杏提取液和高糖培养基IMDM 诱导hUC-MSCs 向胰岛素分泌细胞分化。在hUC-MSCs 诱导前后，用倒置显微镜观察其形态变化，RT-PCR 检测其胰岛相关基因的表达；双硫腙染色鉴定胰岛样细胞团(islet-like clusters, ILCs)；细胞免疫荧光染色检测ILCs 中PDX-1 和免疫活性胰岛素(immunoreactiveinsulin, IRI)的表达；化学发光法检测ILCs 的IRI 分泌量；Western blot 鉴定IRI 的性质。结果显示：纯化的hUC-MSCs 呈间充质干细胞特有的形态特征：长梭形，平行或螺旋形排列；在上述单纯生物学制剂的诱导下，hUC-MSCs 逐渐变圆并聚集成团；在25 cm2 培养瓶的细胞生长面可见上百个ILCs；ILCs 表达胰岛特异性基因pdx-1、insulin；ILCs 呈PDX-1 和IRI 免疫染色阳性反应，双硫腙染色呈阳性；ILCs 可分泌IRI，但多为胰岛素原(proinsulin, PI)。以上结果提示，用表皮生长因子、碱性成纤维生长因子、银杏提取液和高糖培养基IMDM 可诱导hUC-MSCs 快速分化为胰岛素分泌细胞，但ILCs功能不够成熟，难以产生足量真胰岛素。

关键词： 脐带; 间充质干细胞; 胰岛素分泌细胞; 细胞分化; 生物学制剂

分类号：R329

[Rapid differentiation of human umbilical cord-derived mesenchymal stem cellsinto insulin-secreting cells under the sole induction of biological products.] [Ariticle in Chinese]

WANG Yue-Chun, ZHANG Yuan, DUAN A-Lin, LIN Wei-Xia, ZHENG Qiao-Dan, XU Wen-Lu

Department of Physiology； Institute of Hematology, Jinan University Medical College, Guangzhou 510632, China

Abstract

In order to explore the feasibility of inducing the human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) todifferentiate into insulin-secreting cells with biological products alone, hUC-MSCs were separated and purified from the wholeumbilical cord by the sequent digestion of collagenase II and trypsin followed by two-step centrifugation. hUC-MSCs were inducedwith IMDM culture medium containing epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and Ginkgo bilobaextract (GBE). Before and after the induction, the morphological changes were observed under inverse microscope; the islet-relatedgenes were detected by RT-PCR; islet-like clusters (ILCs) were identified by dithizone (DTZ) staining; PDX-1 and immunoreactiveinsulin (IRI) were examined by immunofluorescence method; the quantity and quality of IRI secretion were assayed by chemiluminescenceimmunoassay and Western blot respectively. The results showed that the purified hUC-MSCs presented long spindle-like shapeand parallel or spiral arrangement which are typical morphological features of MSCs. After the induction, hUC-MSCs changedgradually into round or oval shape and gathered together to form ILCs; there were more than one hundred clusters on the growth surfaceof a flask of T25; ILCs were stained into positive mauve by DTZ and positive for PDX-1 and IRI; Western blot displayed that mostof the IRI was proinsulin (PI). Therefore, hUC-MSCs can rapidly differentiate into insulin-secreting cells under the sole induction ofEGF, bFGF, GBE and IMDM, but ILCs are not mature enough to produce sufficient true insulin.

Key words: umbilical cord; mesenchymal stem cells; insulin-secreting cells; cell differentiation; biological products

收稿日期：2009-10-19　　录用日期：2009-12-31

通讯作者：王跃春　　E-mail: monkeymei2@yahoo.com.cn

引用本文：

王跃春, 张洹, 段阿林, 林伟霞, 郑乔丹, 徐文璐. 单纯生物学制剂诱导人脐带间充质干细胞向胰岛素分泌细胞快速分化[J]. 生理学报 2010; 62 (1): 73-78.

WANG Yue-Chun, ZHANG Yuan, DUAN A-Lin, LIN Wei-Xia, ZHENG Qiao-Dan, XU Wen-Lu. [Rapid differentiation of human umbilical cord-derived mesenchymal stem cellsinto insulin-secreting cells under the sole induction of biological products.] [Ariticle in Chinese] . Acta Physiol Sin 2010; 62 (1): 73-78 (in Chinese with English abstract).