miRNAs调节脐带间充质干细胞向内皮细胞分化的研究

Regulation of miRNA for Differentiation of Umbilical Cord Mesenchymal Stem Cells into Endothelial Cells

目的了解间充质干细胞(MSC)分化为内皮细胞的可能的调控机制,以促进其在未来临床实践中的应用。研究目标是脐带来源的间充质干细胞分化成内皮细胞的可行性,以及探索miRNAs在其分化过程中如何发挥调控作用。方法从脐带的华氏胶组织中分离出间充质干细胞,并使用分化试剂盒分化成内皮细胞。流式细胞仪用于鉴定内皮细胞的分化标记。利用Western blot和实时PCR(RT-PCR)检测内皮细胞标志物的蛋白质和mRNA表达。使用生物信息学预测调控CD_(31)或von Willebrand(vWF)因子的miRNAs。在MSC分化的不同阶段,使用RT-PCR测量这些miRNAs在细胞中的水平。并使用双荧光素酶报告实验验证了它们靶向CD_(31)或vWF的3'UTR的能力。结果脐带间充质干细胞能够分化成内皮细胞。分化9 d后,miR-26a-5p、miR-24-3p、miR-15b-5p、miR-497-5p、miR-214-3p、miR-195-5p、miR-761、miR-335-5p和miR-16-5p的水平与未分化的MSCs相比有所下调,而miR-15a-5p和miR-195-5p的水平则没有统计学差异。双荧光素酶报告实验证实,miR-26a-5p靶向CD_(31)的3'UTR,miR-761、miR-335-5p和miR-16-5p靶向vWF的3'UTR。结论MiRNAs可能在间充质干细胞分化为内皮细胞的过程中发挥重要作用。

Objective Understanding the possible regulatory mechanisms of mesenchymal stem cells(MSCs)differentiation into endothelial cells might increase endothelial cell production in clinical practice.The goal of this work was to investigate how microRNAs(miRNAs)changed throughout the differentiation of umbilical cord-derived MSCs into endothelial cells.Methods MSCs were isolated from the umbilical cord's Wharton jelly tissue and differentiated into endothelial cells using a differentiation kit.Flow cytometry was used to measure endothelial differentiation.The protein and mRNA expression of endothelium markers was examined by using Western blot test and real-time PCR(RT-PCR).MiRNAs targeting CD_(31) or von Willebrand factor were predicted using bioinformatics(vWF).The levels of these miRNAs in cells were measured using RT-PCR at different stages of MSC differentiation.Finally,five miRNAs were selected and tested for their ability to target the 3'UTR of CD_(31) or vWF using a dual-luciferase reporter assay.Results The umbilical cord MSCs differentiate into endothelial cells.After 9 days of differentiation,the levels of miR-26a-5p,miR-24-3p,miR-15b-5p,miR-497-5p,miR-214-3p,miR-195-5p,miR-761,miR-335-5p,and miR-16-5p were lowered compared to undifferentiated MSCs,whereas the levels of miR-15a-5p and miR-195-5p were not statistically different.Furthermore,a dual-luciferase reporter experiment confirmed that miR-26a-5p targeted CD_(31)'s 3'UTR and that miR-761,miR-335-5p,and miR-16-5p targeted vWF's 3'UTR,but not miR-15a-5p.Conclusion MiRNAs may play an important role in the differentiation of MSCs into endothelial cells.