过表达微小RNA-199b维持人诱导多能干细胞分化的内皮细胞表型的研究

Overexpression of microRNA-199b-5p maintains endothelial cells phenotype derived from human induced pluripotent stem cells

目的过表达人诱导多能干细胞分化的内皮细胞(hiPSC-EC)中微小RNA(miRNA,miR)-199b-5p,探讨miR-199b-5p对hiPSC-EC传代过程中hiPSC-EC表型转换的影响。方法利用流式细胞术鉴定hiPSC-EC第1代(P1)、第5代(P5)和第10代(P10)内皮细胞表面标志CD31和CD34,将hiPSC-EC分为两组,利用慢病毒分别将过表达miR-199b-5p质粒和空白对照质粒转染hiPSC-EC,实时荧光定量聚合酶链反应(FQ-PCR)检测过表达miR-199b-5p组和空白对照组中miR-199b-5p的表达水平,流式细胞技术鉴定hiPSC-EC过表达组和空白对照组P10细胞表面标志CD31和CD34。应用GraphPadPrism7统计软件分析,数据以均值±标准差(Mean±SD)表示,采用t检验分析过表达组和对照组miR-199b-5p的转录水平和成管数目。结果流式细胞技术检测发现hiPSC-EC在P1~P10传代过程中内皮细胞表面标志CD31和CD34逐渐下降,P188.69%CD31^+/69.57%CD34^+,P568.84%CD31^+/36.90%CD34^+,P1041.66%CD31^+/19.29%CD34^+,FQ-PCR显示miR-199b-5p在hiPSC-ECP1~P10传代过程中显著下降(F=18.190,P<0.01),差异有统计学意义。利用慢病毒载体实现hiPSC-EC过表达miR-199b-5p后,hiPSC-EC在传代过程中内皮细胞表型转换显著被抑制,miR-199b-5p过表达组P1074.33%CD31^+/58.56%CD34^+,对照组P1046.30%CD31^+/38.07%CD34^+。结论miR-199b-5p在hiPSC-EC传代过程中显著下降,miR-199b-5p过表达能够维持传代过程中hiPSC-EC的表型。

Objective MicroRNA-199b-5p (miRNA, miR-199b-5p) overexpression was used to determine the effect of miR-199b-5p overexpression on human induced pluripotent stem cells derived endothelial cell (hiPSC-EC) transdifferentiation. Methods Flow cytometry was used to determine endothelial cell markers (CD31^+, CD34^+) during hiPSC-EC passges at various time points (P1, P5, P10). The expression level of miR-199b-5p was analyzed by real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) and the endothelial cell markers was measured by flow cytometry at passge 10 after miR-199b-5p overexpression and control lentivirus transduction. Results During passages, hiPSC-EC was detected to have a decreased expression level of CD31and CD34 by flowcytometry, P1 88.69% CD31+/69.57% CD34^+, P5 68.84% CD31^+/36.90% CD34^+, P10 41.66% CD31^+/19.29% CD34^+ and the expression of miR-199b-5p was significantly decreased, hiPSC-ECs overexpressing miR-199b-5p maintained endothelial cell phenotype by demonstrating an increased expression level of CD31 and CD34 as compared to control, miR-199b-5p overexpressing iPSC-ECs P10 74.33% CD31^+/58.56% CD34^+ versus control P10 46.30% CD31^+/38.07% CD34^+(F=18.190, P<0.01). Conclusion The expression level of miR-199b-5p was significantly decreased during hiPSC-EC passges and miR-199b-5p overexpression inhibits cell phenotype transition of hiPSCs derived endothelial cells. MiR-199b-5p plays an inhibitory role in iPSC-ECs phenotype transition.