Notch信号通路对VEGF促大鼠间充质干细胞增殖的作用

Effect of Notch Signaling Pathway on VEGF Promoting Rat Mesenchymal Stem Cell Proliferation

本研究旨在探讨Notch信号传导通路在VEGF促大鼠间充质干细胞增殖中的作用。体外培养大鼠间充质干细胞,取对数生长期的细胞用于实验研究。用Notch通路阻断剂DAPT阻断Notch信号传导,观察该通路在VEGF作用下大鼠间充质干细胞的增殖情况。实验分为4组:空白对照组、VEFG组、DAPT组及VEGF+DAPT组。应用CCK-8法检测各组细胞增殖情况,RT-PCR法检测各组相关基因(Notch1、Notch2、Flk-1、HES-1)mRNA水平的变化。结果表明,细胞培养3 d,各时间点(24 h,48 h,72 h)DAPT组及VEGF+DAPT组细胞存活率均较低,细胞形态变圆脱落,数目明显减少;VEGF组存活率最高,差异具有统计学意义(P<0.01);与对照组相比,VEGF组的Notch1、Notch2和Flk-1的表达水平均上调,而DAPT组及VEGF+DAPT组Notch1和Notch2的表达水平均下调,差异均具有统计学意义(P<0.05);VEGF组Hes-1基因水平下调,而DAPT组及VEGF+DAPT组Hes-1基因水平上调,差异具有统计学意义(P<0.05);DAPT组及VEGF+DAPT组的Flk-1基因表达水平稍有下调,但差异无统计学意义(P>0.05)。结论:Notch信号通路在VEGF作用下对大鼠间充质干细胞的增殖具有明显促进作用,而DAPT可抑制这种增殖效应。

This study was purposed to investigate the effect of Notch signaling pathway on VEGF promoting the proliferation of rat mesenchymal stem cells（MSC）. Rat MSC were cultured in vitro, and the cells in logarithmic growth phase were used for experiments. The inhibitor DAPT was used to block Notch signaling pathway, and the effect of the pathway on VEGF promoting proliferation of MSC was observed. The experiment was divided into 4 groups ： control, VEGF, DAPT and VEGF ＋ DAPT. The CCK-8 was used to assay the cells proliferation of each group, while RT-PCR was used to detect the changes of related genes （Notch1, Notch2, Flk-1, Hes-1 ） at mRNA levels. The results indicated that the cells survival rate MSC in DAPT group and VEGF ＋ DAPT group was low in each time point （24 h, 48 h, 72 h）, the cell number decreased, and the cells became rohnded. The survival rate of MSC in VEGF group was the highest; the difference of cell survival rate was statistically significant between the groups （P 〈 0.01 ） ; Compared with the control group, the mRNA expression level of Notchl, Notch2 and Flk-1 in VEGF group was raised, while the expression level of Notchl and Notch2 in DAPT group and VEGF ＋ DAPT group come down, with statistically significant differences （ P 〈 0.05 ） ; whereas the mRNA expression level of Hes-I in VEGF group was down-regulated, but that in DAPT group and VEGF ＋ DAPT group was up-regulated, and the difference was statistically significant （ P 〈 0.05）. Flk-1 mRNA level in DAPT group and VEGF ＋ DAPT group was slightly lower, but the difference was not statistically significant （ P 〉 0.05 ）. It is concluded that Notch signaling pathway plays an important role in promoting the proliferation of rat MSC, treated with VEGF, however, the DAPT can weaken this effect.