miR322-5p干扰对小鼠Sca-1+心脏内皮祖细胞凋亡及分化的影响

Effects of miR322-5p interference on apoptosis and differentiation of mouse Sca-1+cardiac endothelial progenitor cells

目的探讨miR322-5p干扰对小鼠Sca-1+心脏内皮祖细胞凋亡及分化的影响。方法采用流式细胞分选仪分选小鼠CD45-Sca-1+CD31+与CD45-Sca-1+CD31-心脏内皮祖细胞,利用RNA干扰技术(RNAi)慢病毒转染,修饰miR322-5p基因在小鼠Sca-1+心脏内皮祖细胞中的表达,real-time PCR检测miR322-5p基因在小鼠Sca-1+心脏内皮祖细胞中的RNA表达,流式分析miR322-5p干扰后小鼠Sca-1+心脏内皮祖细胞的凋亡情况,免疫荧光观察miR322-5p干扰后小鼠Sca-1+心脏内皮祖细胞向心肌细胞、平滑肌细胞以及血管内皮细胞的分化情况。结果CD45-Sca-1+CD31+、CD45-Sca-1+CD31-细胞占总细胞的比例分别为(3.53±0.47)%、(0.61±0.11)%,两者相比P<0.01;miR322-5p在CD45-Sca-1+CD31+、CD45-Sca-1+CD31-细胞中的相对表达量分别为1.00±0.03、0.04±0.00,两者相比P<0.01;慢病毒转染后,在miR322-5p基因敲低的CD45-Sca-1+CD31+细胞中,miR322-5p相对表达量为0.09±0.02;在miR322-5p基因过表达的CD45-Sca-1+CD31-细胞中,miR322-5p相对表达量为19.33±4.52。CD45-Sca-1+CD31+细胞凋亡率为0.760±0.114,miR322-5p基因被敲低的CD45-Sca-1+CD31+细胞凋亡率为0.556±0.116,两者相比P<0.05。CD45-Sca-1+CD31-细胞凋亡率为0.830±0.013,miR322-5P基因过表达的CD45-Sca-1+CD31-细胞凋亡率为0.903±0.017,两者相比P<0.05。miR322-5p基因被敲低后,CD45-Sca-1+CD31+细胞分化为血管内皮细胞的能力增强。miR322-5p基因过表达后,CD45-Sca-1+CD31-分化为心肌细胞的能力增强,分化为血管内皮细胞的能力降低。结论miR322-5P基因可能是控制CD45-Sca-1+CD31+与CD45-Sca-1+CD31-心脏内皮祖细胞凋亡与分化的一个关键因子。

Objective To explore the effects of miR322-5p interference on the apoptosis and differentiation of mouse Sca-1+cardiac endothelial progenitor cells.Methods Flow cytometer was used to sort mouse CD45-Sca-1+CD31+and CD45-Sca-1+CD31-cardiac endothelial progenitor cells.Using RNA interference(RNA interference,RNAi)lentiviral transfection to modify the expression of miR322-5p gene in mouse Sca-1+cardiac endothelial progenitor cells,real-time PCR was used to analyze the RNA expression level of miR322-5p in mouse Sca-1+cardiac endothelial progenitor cells.Flow cytometry was used to analyze the apoptosis of mouse Sca-1+cardiac endothelial progenitor cells after miR322-5p interference.Immunofluorescence was used to observe the differentiation of mouse Sca-1+cardiac endothelial progenitor cells into cardiomyocytes,smooth muscle cells,and vascular endothelial cells after miR322-5p interference.Results The proportions of CD45-Sca-1+CD31+and CD45-Sca-1+CD31-cells in the total cells were 3.53%±0.47% and 0.61% ±0.11%,respectively,with statistically significant difference(P<0.01);the relative expression levels of miR322-5p in CD45-Sca-1+CD31+and CD45-Sca-1+CD31-cells were 1.0±0.03 and 0.04±0.00,respectively,with statistically significant difference(P<0.01).After lentivirus transfection,the relative expression of miR322-5p in CD45-Sca-1+CD31+cells after miR322-5p gene was knocked down was 0.09±0.02;the relative expression level of miR322-5p in the CD45-Sca-1+CD31-cells with over-expression of miR322-5p gene was 19.33±4.52.The apoptosis rate of CD45-Sca-1+CD31+cells was 0.760±0.114,and the apoptosis rate of CD45-Sca-1+CD31+cells after miR322-5p gene was knocked down was 0.556±0.116,with statistically significant difference(P<0.05).The apoptotic rate of CD45-Sca-1+CD31-cells was 0.830±0.013,and the apoptotic rate of CD45-Sca-1+CD31-cells with over-expression of miR322-5 P gene was0.903±0.017,with statistically significant difference(P<0.05).After the miR322-5p gene was knocked down,the ability of CD45-Sca-1+CD31+cells to differentiate into vascular endothelial cells was enhanced;after the miR322-5p gene was overexpressed,the ability of CD45-Sca-1+CD31-cells to differentiate into cardiomyocytes was enhanced,but the ability to differentiate into vascular endothelial cells was reduced.Conclusion MiR322-5P gene may be a key factor controlling the apoptosis and differentiation of CD45-Sca-1+CD31+and CD45-Sca-1+CD31-cardiac endothelial progenitor cells.