下调胃癌细胞PDGF-D表达对共培养HUVECs生长和功能的影响

Effects of down-regulating expression of PDGF-D in human gastric carcinoma cells on the growth and function of co-cultured HUVECs

目的:探讨通过RNA干扰下调血小板源性生长因子-D(PDGF-D)基因在胃癌SGC-7901细胞中的表达对与其共培养的脐静脉血管内皮细胞(HUVECs)生长和功能的影响.方法:构建靶向PDGF-D的短发夹状RNA(shRNA)真核表达载体,转染胃癌SGC-7901细胞后,通过RT-PCR,Western Blot检测干扰前后PDGF-D的表达;用ELISA法检测培养液上清中血管内皮生长因子(VEGF)的含量;将胃癌细胞SGC-7901与HUVECs共培养后,细胞计数法检测细胞生长变化,流式细胞术测量细胞周期变化,血管形成实验检测HUVECs的血管形成能力.结果:PDGF-D-shRNA1,PDGF-D-shRNA2重组载体均能使SGC-7901细胞中PDGF-D基因在mRNA和蛋白水平上的表达明显下调(P<0.05),且后者抑制效果更好;干扰后胃癌细胞培养液上清中的VEGF下降;与转染后的SGC-7901细胞共培养的HUVECs生长速度明显变慢,且无管状结构形成.结论:重组真核载体能有效抑制SGC-7901细胞的PDGF-D表达,并通过减少VEGF的表达来抑制HUVECs生长和血管形成能力.

AIM：To study the effect of down-regulating expression of PDGF-D in human gastric carcinoma cell line SGC-7901 by RNA interference on the growth and function of co-cultured human umbilical vein endothelial cells（HUVECs）.METHODS：The short hairpinRNA（shRNA） eukaryotic expression vectors targeting PDGF-D were constructed and transfected into SGC-7901 cells.The changes of PDGF-D expression were detected by reverse transcriptase-polymerase chain reaction（RT-PCR） and Western Blot.The concentration of VEGF in supernate was tested by enzyme linked immunosorbent assay（ELISA）.After the co-culture of SGC-7901 cells and HUVECs,the changes of cell growth,cell cycle,and angiogenic ability of HUVECs in vitro were detected by cell counting,flow cytometry,and matrigel in vitro HUVECs tube formation assay,respectively.RESULTS：The eukaryotic expression vectors of PDGF-D-shRNA1 and PDGF-D-shRNA2 could effectively inhibit PDGF-D expression in human gastric carcinoma cell line SGC-7901,and the latter was better（P〈0.05）.The concentration of VEGF in supernate decreased after RNA interference.The proliferation of HUVECs was inhibited after co-cultured with SGC-7901 cells,HUVECs could not form capillary tube-like structures.CONCLUSION：The eukaryotic expression vectors could effectively inhibit PDGF-D expression in human gastric carcinoma cell line SGC-7901,and inhibit the growth of HUVECs and the ability of capillary tube-like structure formation through decreasing VEGF expression.