Mer基因过表达对内皮细胞株HMEC-1细胞血管形成的影响及其机制探讨  

Effect of Mer overepression on HMEC-1 cell angiogenesis and its mechanism

作　　者：范磊[1] 周梦云[1] 沈飞[1] 谢丽倩[1] 阮长耿[1] 

机构地区：[1]苏州大学附属第一医院,江苏省血液研究所,215006

出　　处：《中华血液学杂志》2007年第9期602-604,共3页Chinese Journal of Hematology

基　　金：江苏省卫生厅135重点学科开放课题(135XY0603)

摘　　要：目的探讨受体酪氨酸家族成员 Mer 基因对体外血管形成过程的影响及其具体作用机制。方法利用脂质体法将人全长 Mer 基因真核表达质粒转染到内皮细胞株 HMEC-1细胞中,G418筛选转染细胞得到阳性克隆,利用 PCR 和 Western blot 分别在 mRNA 和蛋白水平验证转染情况。利用Transwell 和 Matrigel 分别观察 Mer 基因过表达对于内皮细胞迁移能力和血管形成能力的影响。通过荧光定量 PCR 筛选血管内皮生长因子(VEGF)-A、VEGF-B、VEGF-C、VEGF-D以及血管内皮生长因子受体(VEGFR)-1、VEGFR-2基因的表达情况。结果经过 G418筛选后,PCR 和 Western blot 检测结果显示 Mer 基因在阳性克隆细胞中有较高水平表达,分别较空质粒对照组上升3.61倍和2.12倍;高表达 Mer 的 HMEC-1细胞迁移能力[迁移到 Transwell 下室壁的细胞为(21±6)/视野]较对照组[(36±11)/视野]明显下降。体外血管形成实验也表明高表达 Mer 基因的 HMEC-1细胞血管形成能力明显下降,抑制率为76.9%;荧光定量 PCR 结果显示 VEGF-C 和 VEGFR-2表达明显下降,VEGF-C 表达量为对照组的44.7%,VEGF-C 表达为对照组的25.6%。结论 Mer 过表达可以显著抑制 HEMC-1细胞的迁移能力和血管形成能力,并且可能是通过 VEGF-C/VEGFR-2信号途径发挥作用。Objective To explore anti-angiogenesis effect of Mer, a member of tyrosine kinase receptor family, and its mechanism. Methods Human Mer full length plasmid was transfected into HMEC-1 cells through liposome. G418 was used to select positive clone. Expression of Mer at mRNA and protein level was detected by real-time PCR and Western-blot, respectively. Transwell and Matrigel were used to evaluate the effect of overexpressed Mer on migration and angiogenesis of HMEC-1 cells. Primary angiogenesis associ- ated factor VEGF-A, VEGF-B, VEGF-C, VEGF-D and VEGFR-1, VEGFR-2 were screened by real-time PCR. Results After G418 selection, the Met expression in transfected HMEC-1 cells was increased 3.61- and 2.12 fold at mRNA and protein level, respectively. Compared with negative control, the migration of Mer-HMEC-1 was decreased （21± 6 vs 36 ± 11 ）, and angiogenesis capability on Matrigel significantly decreased. By real-time PCR, the expreesion of VEGF-C and VEGFR-2 was down-regulated to 44.7% and 25.6% of the negative control. Conclusion Overexpressed Mer tyrosine kinase receptor can inhibit the mi- gration and angiogenesis of HMEC-1 cells through VEGF-C/VEGFR-2 signal pathway.

关 键 词：基因 Mer 受体酪氨酸激酶 血管新生 细胞系 HMEC-1 内皮生长因子 受体 内皮生长因子 

分 类 号：R543[医药卫生—心血管疾病]