【摘要】 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。更多还原

【Abstract】 Objective Design the cell-penetrating peptide TAT-DV3 to deliver Bcl-2 siRNA,and study its effect on glioma U251 cells in vitro. Methods Design and synthesize Bcl-2 siRNA,and assemble them into TAT-DV3. U251 cells were cultured in vitro,and then treated with TAT-DV3-Bcl-2 siRNA. The intracellular distribution of siRNA was detected by confocal laser scanning microscopy. The expression of Bcl-2 mRNA and protein were detected by q PCR and Western blotting respectively. The percentages of apoptotic U251 cells were detected by flow cytometry. Results Laser Scanning Confocal Microscope indicated that TAT-DV3-Bcl-2 siRNA was successfully delivered into cells. The results of PCR and Western blotting showed that the expression of Bcl-2 was reduced in the group of TAT-DV3-Bcl-2 siRNA. Flow Cytometry revealed TAT-DV3-Bcl-2 siRNA could induce U251 cell apoptosis and death. Conclusion TAT-DV3 could deliver Bcl-2 siRNA into U251 cells,significantly reduced the expression of Bcl-2 mRNA and protein,and caused U251 cell apoptosis obviously.更多还原