摘要
目的观察RNA干扰技术沉默Cyt61基因表达对人脑胶质瘤U251细胞生物学行为的影响。方法针对Cyr61 mRNA的序列设计合成小干扰RNA（siRNA）的DNA模板，构建pR-NAT-Cyt61重组质粒，转染人脑胶质瘤U251细胞；RT-PCR和Westem blot检测其对U251细胞内源性Cyt61表达的影响；用噻唑蓝（MTT）比色法观察U251细胞体外增殖活性的变化；用Transwell小室法检测U251细胞体外侵袭能力的改变；流式细胞仪检测U251细胞凋亡并用透射电镜观察凋亡后的细胞形态学变化。结果pRNAT-Cyt61重组质粒在mRNA及蛋白水平分别显著抑制Cyt61基因表达，抑制率分别最高达到74．87％和78．23％；U251细胞的体外生长抑制率最高达68．15％，侵袭细胞数下降至（46．00±2．82）个；U251细胞凋亡率最高达53．16％。结论pRNAT-Cyt61可抑制Cyr61在人脑胶质瘤U251细胞中的表达，并抑制细胞的增殖活性和侵袭能力，促进细胞凋亡。
Objective To explore the relationship between the expression of Cyr61 and the biological behaviors of human glioma U251 cells by decreasing Cyr61 gene expression by RNA interference. Methods One pair of DNA template coding was synthesized against U251 to reconstruct pRNA-Cyr61, which was transfected into U251 cells. The Cyr61 expression in U251 cells transfected with pRNAT-Cyr61 was detected by RT-PCR and Western blot. MTT,Transwell and FCM were used to observe the biological behaviors of U251 cells. Transmission electron microscopy （ TEM ） was applied to observe the changes of cell morphology after Cyr61 siRNA transfection. Results RT-PCR and Western blot analyses demonstrated that pRNAT-Cyr61 could significantly inhibit the expression of Cyr61 to 74.87% and 78.23% respectively in U251 cells; MTF, Transwell and FCM results showed that pRNAT-Cyr61 could significantly inhibit the proliferation by 61.83% and reduce the invasive cell number to （46.00 ± 2.82） respectively. The occurrence of apoptosis was increased significantly to 53.16% in U251 cells in vitro. Conclusion pRNAT-Cyr61 could significantly inhibit the expression of Cyr61, suppress the growth and invasion of U251 ceils and induce apoptosis.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2008年第4期477-479,共3页
Chinese Journal of Experimental Surgery
