CCN3对骨髓间充质干细胞向内皮细胞分化的影响

Influence of CCN3 on differentiation of bone marrow mesenchymal stem cells into endothelial cells

目的:探讨肾母细胞瘤过度表达基因(CCN3)对骨髓间充质干细胞(BM-MSCs)向内皮细胞分化的影响。方法:以含50ng/ml血管内皮生长因子培养基诱导BM-MSCs向内皮细胞分化作为阳性对照组,以含100ng/ml重组CCN3蛋白并50ng/ml血管内皮生长因子培养基诱导BM-MSCs向内皮细胞分化作为CCN3组,并以单纯完全培养基培养BM-MSCs为阴性对照组,采用细胞免疫荧光化学法和半定量逆转录聚合酶链反应法(RT-PCR)检查诱导16d后假性血友病因子(vWF)表达以评价内皮细胞分化,以半定量RT-PCR法检测BM-MSCs诱导分化前后Notch1基因mRNA表达。结果:BM-MSCs诱导分化16d后,阳性对照组可见部分细胞vWF荧光染色阳性,CCN3组阳性染色细胞明显多于阳性对照组,且vWF mRNA表达明显强于阳性对照组[吸光度(OD)值比:(0.550±0.090)比(0.358±0.080),P<0.01],阴性对照组未见阳性染色细胞;此外,诱导分化前,三组Notch1基因mRNA均呈低表达,组间两两比较无明显差异(P均>0.05),诱导分化16d后阳性对照组和CCN3组Notch1基因mRNA表达明显强于阴性对照组[OD值比:(0.232±0.047)、(0.352±0.029)比(0.132±0.033),P均<0.01],但与阳性对照组相比,CCN3组Notch1基因mRNA表达明显更高(P<0.01)。结论:肾母细胞瘤过度表达基因通过激活Notch1信号通路可增强骨髓间充质干细胞向内皮细胞分化的能力。

Objective：To explore the influence of nephroblastoma over expressed gene （CCN3）on differentiation of bone marrow mesenchymal stem cells （BM-MSCs）into endothelial cells.Methods：BM-MSCs differentiation into endothelial cells induced by condition medium containing vascular endothelial growth factor （VEGF,50ng/ml）was regarded as positive control group.BM-MSCs differentiation into endothelial cells induced by condition medium containing recombined CCN3 protein （100 ng/ml）and VEGF （50 ng/ml）was regarded as CCN3 group,and BM-MSCs incubated in pure complete medium was regarded as negative control group.Immunofluorescence staining and semiquantitative reverse transcription-polymerase chain reaction method （RT-PCR）were used to measure expression of von Willebrand factor （vWF）after 16d to evaluate endothelial cell differentiation,and semiquantitative RT-PCR method was used to measured expression of Notch1 gene mRNA before and after induced BM-MSCs differentiation. Results：On 16d after induced BM-MSCs differentiation,some cells of positive vWF fluorescence staining were observed in positive control group,those of CCN3 group was significantly more than those of positive control group, and expression of vWF mRNA [ratio of optical density （OD）： （0.550±0.090）]of CCN3group was significantly higher than that of positive control group （0.358±0.080）,（P〈0.01）,and no vWF-positive cells were observed in＆amp;nbsp;negative control group;Besides,before induced differentiation,expression of Notch1 gene mRNA were low in three groups without significant difference between any two groups （P＆gt;0.05 all）,on 16d after induced differentiation, expressions of Notch1 gene mRNA in positive control group （0.232±0.047）and CCN3 group （0.352±0.029）were significantly higher than that of negative control group （0.132±0.033）,P〈0.01 all,it of CCN3 group was significantly higher that of positive control group （P〈0.01）.Conclusion：CCN3 can enhance BM-BMCs differentiation into endothelial cells by activating Notch1 signal pathway.