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目的探讨人脐静脉内皮细胞株(HUVEC)中类表皮生长因子功能域7(EGFL7)对共培养的人主动脉平滑肌细胞(AoSMC)凋亡的影响以及可能的信号转导通路。方法利用细胞共培养池行HUVEC与AoSMC共培养,将EGFL7的特异siRNA转染HUVEC细胞,利用MTS法检测HUVEC中EGFL7基因的有效沉默对共培养的AoSMC细胞存活率的影响;并合成无关siRNA作为阴性对照组(neg组),以未转染siRNA的细胞为空白对照组(con组)。同时利用分光光度法检测其对AoSMC细胞乳酸脱氢酶(LDH)、三磷酸腺苷(ATP)以及细胞凋亡蛋白酶(Caspase-3)表达的影响;应用RT-PCR检测共培养AoSMC的bcl-2及bax mRNA表达水平的改变。结果与neg组及con组相比,干扰HUVEC细胞EGFL7表达12 h后,AoSMC存活率无明显变化(P>0.05),而干扰24、36、48 h后,存活率显著降低(P<0.05);干扰12、24、36、48 h后,AoSMC细胞线粒体ATP释放量减少、培养基中LDH含量及Caspase-3表达量均增加(P<0.05)。RT-PCR检测结果显示,与neg组及con组相比,干扰siRNA转染HUVEC细胞24、36、48 h,共培养的AoSMC凋亡基因bcl-2表达水平轻度降低(P<0.05);但Bax表达水平在相应干预时间点均无明显改变(P>0.05)。结论内皮细胞株EGFL7基因沉默时,可导致平滑肌细胞凋亡;EG-FL7通过bcl-2相关信号通路调节SMC凋亡,而与Bax基因表达无关。
Objective To investigate the effect of epidermal growth factor-like domain 7 (EGFL7) on the apoptosis of human aortic smooth muscle cells (AoSMC) cultured in human umbilical vein endothelial cell line (HUVEC) and its possible signal transduction pathway. Methods HUVECs were co-cultured with AoSMC in cell co-culture well and EGFR7 specific siRNA was transfected into HUVEC cells. MTS assay was used to detect the effect of EGFR7 gene silencing on the survival rate of co-cultured AoSMC cells. The negative control group (neg group) and the untransfected cells were blank control group (con group). At the same time, the effect of LDH, ATP and Caspase-3 on AoSMC cells was detected by spectrophotometry. The bcl-2 and bax Changes in mRNA expression levels. Results Compared with the neg and con groups, the survival rate of AoSMC was not significantly changed after interfering with the expression of EGFL7 in HUVECs for 12 h (P> 0.05), but significantly decreased after 24, 36 and 48 h (P <0.05) ; After 12, 24, 36 and 48 h of interference, mitochondrial ATP release in AoSMC cells decreased, and the content of LDH and Caspase-3 in the medium increased (P <0.05). The results of RT-PCR showed that compared with the neg and con groups, the expression level of bcl-2 in the co-cultured AoSMCs was slightly decreased (P <0.05) after the siRNAs were transfected into HUVECs for 24, 36 and 48 h. However, the expression level of Bax had no significant change at the corresponding time points (P> 0.05). CONCLUSION: EGFL7 gene silenced in endothelial cells can induce apoptosis of smooth muscle cells. EG-FL7 regulates SMC apoptosis through bcl-2 related signaling pathway, but not with Bax gene expression.