摘要
目的为探索治疗白血病的新思路研究了小干扰RNA(siRNA)对肿瘤细胞株HL60和人血管内皮细胞(EC)株EA.hy926血管内皮细胞生长因子受体2(VEGFR2)基因表达的抑制作用。方法制备并筛选VEGFR2基因的最有效siRNA,并据此设计shRNA寡核苷酸链,构建pENTRTM/U6干扰表达载体。瞬时转染细胞,采用MTT法、RT-PCR法及Westernblot测定VEGFR2表达抑制情况。结果VEGFR2siRNAa、b、c抑制HL60细胞效率分别为77.5%、45.0%、80.9%,以siRNAc最高,利用其shRNA和pENTRTM/U6构建的入门克隆转染HL60,抑制细胞效率达99.1%;此入门克隆对VEGFR2基因mRNA和蛋白的表达抑制在HL60和EA.hy926细胞均显著。结论在体外shRNA表达载体可有效抑制HL60细胞VEGFR2自分泌和旁分泌,有效抑制白血病细胞生长。
[Objective] To explore a new way to treat leukemia, the inhibitory effect of small interfering RNA （siRNA） on VEGFR2 gene expression of tumor cell line HL60 and human endothelial cell （EC） line EA·hy926 was studied. [Methods] siRNAs for VEGFR2 gene were designed and the most efficient was chosen after being transfected into HL60 cells, pENTR^TM/U6 interfering expression system was constructed. HL60 and EA. hy 926 cells were transfected. VEGFR2 expression was tested with MTT assay, RT-PCR, and Western blot analysis. [Results]The inhibitory rates of VEGFR2 siRNA a, b, c were 77.5% ,45.0% ,80.9% respectively,among which of siRNAc being the highest. VEGFR2 expression in HL60 and EA. hy926 was beth inhibited by using shRNA and pENTR^TM/U6 entry clone. For HL60 cell,the inhibitory rate was 99.1%.[Conclusion] shRNA interfering expression system can inhibit VEGFR2 self-secretion and para-secretion of HL60 cell in vitro, which inhibits leukemia growth.
出处
《中山大学学报（医学科学版）》
CAS
CSCD
北大核心
2006年第3期276-280,共5页
Journal of Sun Yat-Sen University:Medical Sciences
基金
广东省自然科学基金资助项目(5100992)
