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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 strategy to induce differentiation of human embryonic stem cells (ESC) into dendritic cells and to explore the role of miR-223 in the regulation of human embryonic stem cell-derived dendritic cells and its molecular mechanisms. Methods: Adherent human embryonic stem cells were directly adhered to the extracellular matrix of type Ⅳ collagen. Hematopoietic growth factors were added to the hematopoietic stem / progenitor cells, myeloid progenitor cells and dendritic cells in a stepwise manner. The morphological, Flow cytometry and hematopoietic colony formation assay identified differentiated cells; human embryonic stem cells were transfected with lentivirus overexpression of miR-223 or inhibition of its expression started to dendritic cells differentiated compared miR-223 expression levels differentiated cells hematopoietic The number of colony formation and the expression of surface markers; using dual luciferase reporter gene assay confirmed that TGFBR3 is a direct target miR-223 play a role. Results: The human ES cells were successfully induced to dendritic cells. The proportion of CD83 in differentiated cells was up to 82%. The expression of miR-223 was up-regulated in the whole differentiation process. The expression of CD34 + CD45 CD34 ~ + CD45 ~ + and CD83 ~ + were significantly higher than those of the miR-223 inhibitor group and the negative control group (P <0.05). The cells labeled with miR-223 inhibitor (P <0.05). The numbers of about 759 CFU / 105 cells in the group of miR-223 mimic cells were significantly higher than those in other groups (P <0.05). The dual luciferase reporter gene The results showed that miR-223 significantly inhibited the luciferase activity (decreased by 37%) of TGFBR3-3’UTR structure (P <0.05), and the luciferase activity of TGFBR3-3’UTR mutant was significantly higher than that of wild-type P <0.01). With the differentiation and maturation of dendritic cells, the expression levels of TGFBR3 in the differentiated cells supplemented with miR-223 mimics decreased gradually, whereas the addition of miR-223 inhibitor significantly increased TGFBR3 due to the inhibition of endogenous miR-223 expression. Conclusion: miR-223 regulates the differentiation of human embryonic stem cells into dendritic cells, which may promote the generation of embryonic stem cell-derived dendritic cells by acting directly on the target TGFBR3.