摘要
目的:建立人胚胎干细胞(ESC)向树突细胞诱导分化的新策略,探讨microRNA-223(miR-223)在人胚胎干细胞源树突细胞产生过程中的调控作用及其分子机制。方法:采用人胚胎干细胞在胞外基质Ⅳ型胶原蛋白上直接贴壁培养,加入造血生长因子分步向造血干/祖细胞、共同髓系祖细胞和树突细胞诱导的方案,通过形态学、流式细胞术和造血集落形成实验鉴定分化细胞;人胚胎干细胞经慢病毒转染过表达miR-223或抑制其表达后启动向树突细胞分化,比较不同miR-223表达水平下分化细胞中造血集落形成的数目和表面标记物的表达量;应用双荧光素酶报告基因检测验证TGFBR3是miR-223发挥作用的直接靶标。结果:人ES细胞成功诱导为树突细胞,分化细胞中表达CD83比例可达82%,在整个分化过程中miR-223表达呈上调趋势;添加miR-223模拟物组分化细胞表达CD34^+CD45^+、CD34^+CD45^+以及CD83^+的比例均显著高于添加miR-223抑制剂组和阴性对照组(P<0.05);添加miR-223抑制剂组分化细胞表达各阶段细胞标记物显著低于阴性对照组(P<0.05);添加miR-223模拟物组分化细胞出现大约759个CFU/105细胞数,显著高于其它各组(P<0.05);双荧光素酶报告基因结果显示,miR-223显著抑制TGFBR3-3'UTR结构的荧光素酶活性(下降了37%)(P<0.05),而且TGFBR3-3'UTR突变型的荧光素酶活性显著高于野生型(P<0.01);随着向树突细胞分化成熟,添加miR-223模拟物组分化细胞表达TGFBR3水平逐渐下降,而添加miR-223抑制剂组由于内源性miR-223受到抑制而明显上调TGFBR3的表达。结论:miR-223调控人胚胎干细胞向树突细胞分化,可能通过直接作用于靶标TGFBR3而促进胚胎干细胞源树突细胞的产生。
Objective： To establish a new inducing system for differentiation of human embryonic stem（ES） cells to dendritic cells（DC）,and further explore how microRNA-223（miR-223） regulates DC differentiation from ES cells.Methods： Human ES cells were cultured on plates coated by Ⅳ type collagen and differentiated into hematopoietic stem/progenitor cells,common myeloid progenitor cells and DC step by step via adding different hematopoietic growth factors. The differentiated cells were identified by morphology,flow cytometry and hematopoietic colony forming unit（CFU） assays. Human ES cells were transfected with lentiviral vectors to overexpress miR-223 or inhibit miR-223 expression,then initiated the differentiation to DC. The differentiated cells at the different miR-223 levels were compared by the numbers of hematopoietic CFU and the expressions of specific surface markers. Dual-luciferase reporter assay was performed to test whether miR-223 directly targets TGFBR3. Results： Human ES cells were successfully induced into DC as the percentage of CD83 was approximately 82%,and the expression of miR-223 was up-regulated during the whole process. Supplementing miR-223 level using synthetic miR-223 mimics improved the proportions of CD34^＋ CD45^＋ ,CD34^＋ CD45^＋ and CD83^＋ in differentiated cells,which were significantly higher than those in synthetic miR-223 inhibitor group and negative control（P〈0. 05）. The expressions of cell makers at each differentiated phase in miR-223 inhibitor group were significantly lower than those in negative control（P〈0. 05）. The differentiated cells in miR-223 mimics group showed approximately 759 CFUs per 105 cells,which was significantly higher than that in others（P〈0. 05）. Compared with negative control,miR-223 substantially inhibited the luciferase activity of Tgfbr3 3＇ UTR construct（by 37%）（P〈0. 05）. In addition,the luciferase activity of the mutant construct was significantly higher thanthat of the WT construct in the presence of
出处
《中国实验血液学杂志》
CAS
CSCD
北大核心
2017年第5期1275-1282,共8页
Journal of Experimental Hematology
基金
北京市自然基金资助项目(7163229)
高等学校博士学科点专项科研基金资助(20120001120132)
