TAT-DV3-Bcl-2siRNA促进U251胶质瘤细胞凋亡的研究

TAT-DV3-Bcl-2 siRNA induces the U251 cell apoptosis in vitro

目的设计合成肿瘤靶向穿膜肽TAT-DV3,联合Bcl-2 siRNA,体外实验研究其对U251胶质瘤细胞的作用。方法设计合成特异性Bcl-2 siRNA及靶向肽TAT-DV3,利用静电作用使两者结合。体外培养U251胶质瘤细胞,TATDV3-Bcl-2 siRNA复合物处理细胞后,激光共聚焦显微镜追踪Bcl-2 siRNA进入细胞的分布。PCR检测靶基因Bcl-2沉默情况,Western blot检测Bcl-2蛋白表达;Annexin-V FITC与PI联合标记细胞后,流式细胞仪检测细胞凋亡。结果激光共聚焦显微镜结果证明TAT-DV3-Bcl-2 siRNA成功进入U251细胞内。PCR及Western blot验证了TAT-DV3-Bcl-2 siRNA沉默了目标基因Bcl-2。利用流式细胞仪进行凋亡检测,发现与对照组相比TAT-DV3-Bcl-2 siRNA可以显著促进U251细胞凋亡。结论靶向肽TAT-DV3可有效投递Bcl-2 siRNA进入U251胶质瘤细胞,通过降解Bcl-2 mRNA抑制Bcl-2的表达并促进胶质瘤细胞的凋亡。

Objective Design the cell-penetrating peptide TAT-DV3to deliver Bcl-2siRNA,and study its effect on glioma U251cells in vitro.MethodsDesign and synthesize Bcl-2siRNA,and assemble them into TAT-DV3.U251cells were cultured in vitro,and then treated with TAT-DV3-Bcl-2siRNA.The intracellular distribution of siRNA was detected by confocal laser scanning microscopy.The expression of Bcl-2mRNA and protein were detected by qPCR and Western blotting respectively.The percentages of apoptotic U251cells were detected by flow cytometry.ResultsLaser Scanning Confocal Microscope indicated that TAT-DV3-Bcl-2siRNA was successfully delivered into cells.The results of PCR and Western blotting showed that the expression of Bcl-2was reduced in the group of TAT-DV3-Bcl-2siRNA.Flow Cytometry revealed TAT-DV3-Bcl-2siRNA could induce U251cell apoptosis and death.ConclusionTAT-DV3could deliver Bcl-2siRNA into U251cells,significantly reduced the expression of Bcl-2mRNA and protein,and caused U251cell apoptosis obviously.