9R穿膜肽可增强P53抗肿瘤效果  

Anti-tumor efficacy of P53 with 9R cell-penetrating peptides

作　　者：刘源[1] 陈睿[1] 张楠[1] 叶贤龙[1] 白银[1] 魏雨泉[1] 任桂萍[1] 李德山[1] 

机构地区：[1]东北农业大学生命科学学院生物制药教研室,黑龙江哈尔滨150030

出　　处：《生物工程学报》2013年第7期955-964,共10页Chinese Journal of Biotechnology

基　　金：黑龙江省博士后科研启动基金(No.LBH-Q09162);黑龙江省教育厅科学技术研究项目(No.12521z004)资助~~

摘　　要：为解决P53蛋白难以进入细胞内部发挥治疗作用的瓶颈难题。将p53基因融合插入带有9个精氨酸作为穿膜肽的表达载体中表达融合蛋白CPPs-P53,并与没有穿膜肽的P53蛋白进行比较,利用Western blotting方法检测蛋白的表达情况,MTT及Annexin V/PI双染法检测细胞生长抑制率及细胞凋亡率。Western blotting检测表明已成功在原核表达系统中表达融合蛋白CPPs-P53和P53蛋白,且蛋白纯度均已达到90%以上;MTT检测表明,P53蛋白对肿瘤细胞的生长虽有一定的抑制作用,但融合蛋白CPPs-P53与之相比,对肿瘤细胞生长的抑制效果显著增强,细胞生长抑制率有明显的提升,并且细胞生长抑制率呈现剂量依赖性;Annexin V/PI双染检测细胞凋亡情况也表明P53虽可以在一定程度上诱导肿瘤细胞的凋亡,但与P53蛋白相比较,融合蛋白CPPs-P53诱导的凋亡细胞明显增加,凋亡率是P53蛋白的2~3倍。由此说明在抑制肿瘤细胞的生长和诱导细胞凋亡方面,CPPs-P53比没有穿膜肽的P53蛋白的效果更显著。To enhance the penetration of P53 into tumor cells by fusion it with the cell penetrating peptide 9R.The fusion gene of 9R-p53 was cloned into the expression vector.The fusion protein,CPPs-P53,was expressed and purified.We detected the rate of cell growth inhibition and apoptosis by MTT and Annexin-V-FITC/PI double stained method respectively for measuring its effect on tumor cells.CPPs-P53 and P53 were successfully expressed and purified,the purity of both proteins reached up to 90%.MTT assay showed that the cell growth inhibition by CPPs-P53 was more efficient than P53,and the rate of cell growth inhibition is dose-dependent.The apoptosis experiment showed that P53 could induce apoptosis of tumor cells.Compared with the P53,CPPs-P53 had a more significant effect in inducing cell apoptosis（＊＊P0.01）.The CPPs-P53 shows more significant effects than P53 in inhibiting cell growth and inducing apoptosis on tumor cells.

关 键 词：P53 CPPs-P53 穿膜肽 细胞凋亡 

分 类 号：R96[医药卫生—药理学]