人胚胎干细胞诱导分化为血液血管干细胞的条件优化  

Optimization Condition of Hemangioblastic Differentiation from Human Embryonic Stem Cells

作　　者：王建[1] 赵惠萍[1] 林戈[1] 卢光琇[1] 

机构地区：[1]中南大学生殖与干细胞工程研究所人类干细胞国家工程研究中心,湖南长沙410078

出　　处：《实用预防医学》2009年第4期1031-1035,共5页Practical Preventive Medicine

摘　　要：目的探讨人类胚胎干细胞(human embryonic stem cells,hESCs)向血液血管干细胞分化的方法和培养条件。方法人类胚胎干细胞自发分化形成拟胚体(human embyonic bodies,hEBs),与胚胎骨髓基质细胞(human fetal bown marrow stromal cells,hFBMSCs)体外共培养,并添加诱导造血分化的细胞因子如干细胞生长因子(stemcellfactor,SCF)、血管内皮细胞生长因子(vascular endothelial growth factor,VEGF)、血小板生成素(Thrombopoietin,TPO)等因子,诱导分化6d,流式细胞仪进行血液血管干细胞标记KDR+/CD34+的检测,RT-PCR对血液血管干细胞相关基因进行检测,同时对不同浓度的SCF、VEGF、TPO诱导效率进行分析,探讨诱导分化的优化条件,最终形成的条件,再次采用流式细胞学进行验证诱导分化效率的改善。结果hEBs在机制细胞上可以形成血岛样集落,经过消化后,流式检测发现其中KDR+/CD34+细胞比例为4.1%±0.5%,与对照组比较差异有统计学意义。经过RT-PCR检测发现血岛样细胞表达血液血管干细胞相关基因如:人血管内皮生长因子受体2(kinase insert domain protein receptor,KDR)、干细胞白血病因子(Stem Cell Leucamia,SCL)、B细胞特异性白血病病毒插入位点(B-cell-specific moloney leukemia virus insert site 1,Bmi1)、GATA结合蛋白2(GATA binding protein2,Gata2)、runt相关转录因子1(runt-related transcription factor 1,Runx1)、血小板内皮细胞粘附分子(platelet/endothelial cell adhesion molecule,CD31)、内皮细胞特异性酪氨酸激酶受体2(endothelium-specific receptor tyrosine kinase2,Tie-2)。在对不同浓度的干细胞因子进行诱导分化,发现在VEGF(10～40ng/ml),SCF(100～300ng/ml)对血液血管干细胞集落的形成有促进作用,在此范围内集落数量和细胞因子成剂量依赖的正相关关系。优化条件后,诱导效率显著提高,KDR+/CD34+细胞为18.8%±2.5%。结论采用基质细胞联合细胞因子共培养的方法可以将人类胚胎干细胞诱导分化�Objective To establish an optimization method for directing the human embryonic stem cells to differentiate into the hemangioblastic like cell. Methods The 2^nd day hEBs were co- cultured with hFBMSCs supplemented with cyto- kines, such as SCF, VEGF, and TPO. After 6 days induction, the blood island- like cell clusters were isolated and flow cytometric analysis-was performed to detect the cells with hemangioblastic marker, such as KDR^＋/CD34^＋ . Gene expression profile of the induced cell was checked by RT- PCR to evaluate the hemangioblastic related gene expression level, such as KDR, SOL, Bmi, Gata2, Runx1, CD31, and Tie-2. Then different doses of VEGF, SCF, and TPO were added in the medium to optimize the induction condition. The hemangioblastic clusters were counted to analyze the dose dependent relation to each cytokine. Then flow cytometric analysis was performed to test the optimization condition by hemangioblastic cell marker KDR^＋/CD34^＋ . Results After 6 days induction, the hEBs differentiated into kinds of blood-island like cell clusters. The flow cytometric analysis shewed that there were 4.196 ± 0.5 % KDR^＋/CD34^＋ cells in the island - like cells cluster and the RT - PCR results showed that these cells expressed the hemangioblastic related genes such as KDR, SOL, Bmil, Runx1, GA- TA2, Tie2, and CD31. When the island - like cell clusters were counted, the results showed a dose dependent relationship between the number of clusters and VEGF （10ng/ml-40ng/ml） or SCF （100ng/ml-300ng/ml）, but TPO did not have the significant effect. A new round induction using the optimized parameters showed that the ratio of KDR^＋/CD34^＋ cell increased significantly to 18.8 % ±2.5 % by the flow cytometrics analysis. Conclusions hESCs cell could be directed to differentiate into hemangioblastic like cells by co- culturing with hFBMSC supplemented with VEGF an.d SCF, 40ng/mlVEGF and 300ng/ml SCF could enhance the induction effectively.

关 键 词：人类胚胎干细胞 诱导分化 血液血管干细胞 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]