尼克酰胺、β-细胞调节素、bFGF、HGF联合诱导人脐血CD34^+细胞向胰岛素分泌细胞的分化  

THE DIFFERENTIATION OF HUMAN UMBILICAL CORD BLOOD CD34^+ CELL INTO INSULIN-SECRETING CELLS INDUCED BY NICOTINAMIDE,BETACELLULIN,BFGF AND HGF

作　　者：张芳婷[1] 万汇娟[1] 区文超[2] 龙霞[1] 叶静[1] 于洁[1] 鲁树坤[2] 房家智[1] 

机构地区：[1]北京大学深圳医院中心实验室,深圳518036 [2]北京大学深圳医院超声影像科,深圳518036

出　　处：《解剖学报》2007年第2期192-195,共4页Acta Anatomica Sinica

基　　金：深圳市科技计划项目(200404106);广东省自然科学基金资助项目(31005)

摘　　要：目的探讨在体外培养条件下用尼克酰胺、β-细胞调节素(betacellulin)、bFGF、HGF诱导人脐血CD34+细胞向胰岛细胞的分化。方法用磁性细胞分选试剂盒(MACS)分离出CD34+细胞后在含5%FBS,1×ITS,4.7mg/L亚油酸,10-4mol/L 2磷-酸抗坏血酸的低糖型DMEM中培养,扩增后于培养第5d加入尼克酰胺、betacellulin、bFGF、HGF进行诱导,并于诱导14d、24d留取细胞。应用RT-PCR、免疫细胞化学染色方法检测分化细胞中胰岛细胞标志物,并用ELISA方法检测培养液中胰岛素水平。结果流式细胞仪检测结果显示,CD34+细胞的平均分离纯度>90%,达到分离要求。RT-PCR检测到诱导后的CD34+细胞表达nestin、ngn3、IPF-1 mRNA。免疫细胞化学染色可见诱导的CD34+细胞中出现nestin和insulin阳性表达细胞。胰岛素分泌细胞的分化率平均为(9.8±2.7)%。ELISA检测发现诱导组和未诱导组培养液中的insulin含量有显著性差异(P<0.01)。结论体外联合应用上述因子能诱发脐血CD34+造血干细胞向胰岛素分泌细胞的分化。Objective To investigate the differentiation of CD34^＋ cells derived from human umbilical cord blood into insulin-secreating cells in an in vitro system supplemented with growth factors. Methods CD34^＋ cells were isolated by magnetic cell sorting（ MACS）. The purity of acquired cells was determined by FACS analysis. Purified CD34^＋ cells were cultured in DMEM supplemented with 5% FBS, 1 × ITS, 10^-4 mol/L ascorbic acid 2-phosphate, 4.7mg/L linoleic acid, and a combination of nicotinamide, betacellulin, bFGF and HGF. Cultured cells were collected on the 14th and 24th days after the induction to determine the extent of the cell conversion by microscopic observation on morphology, RT-PCR on the transcription of nestin, ngn3 and IPF-1 mRNA, immunocytochemistry on the expression of nestin and insulin, ELISA on the quantification of the insulin product in the medium. Results The isolation purity degree of CD34^＋ cells was greater than 90% after MACS. Nestin, ngn3 and IPF- 1 mRNA were detected in the differentiated cells. Nestin and insulin positive cells were ovserved in the differentiated cells with immumocytochemical staining. The positive ratio of the differentiated cells were （9.8 ± 2.7） % . Insulin ELISA result showed that the insulin product in the culture medium was significantly increased after induction in comparison with that in the control groups （ P 〈 0.01 ）. Conclusion The differentiation of CD34^＋ cells derived from human umbilical cord blood into insulin-secreting cells may be induced by the growth factor mentioned above.

关 键 词：胰岛细胞 分化 酶联免疫吸附测定 免疫细胞化学 脐血CD34^+细胞 

分 类 号：Q254[生物学—细胞生物学]