Zhang Jianxu, Wu Di, Xing Zhen, Liang Shaojun, Han Haobo, Shi Hui, Zhang Yan, Yang Yan, Li Quanshun
Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, School of Life Sciences, Jilin University, Changchun 130012, China.
Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, School of Life Sciences, Jilin University, Changchun 130012, China.
Colloids Surf B Biointerfaces. 2015 May 1;129:54-62. doi: 10.1016/j.colsurfb.2015.03.032. Epub 2015 Mar 18.
In this paper, N-isopropylacrylamide-modified polyethylenimine (PEN) was constructed through Michael addition and employed as a carrier to achieve the p53 gene delivery, using HeLa (p53wt) and PC-3 cells (p53null) as models. After PEN-mediated p53 transfection, expression level of p53 in HeLa and PC3 cells was up-regulated at both mRNA and protein levels. Due to the exogenous p53 expression, the inhibition of cell proliferation was observed through MTT analysis, attributing to the activation of apoptosis and cell cycle arrest. Using flow cytometric analysis, early apoptotic ratios of 54.95% and 27.06% after PEN-mediated p53 transfection were detected in PC-3 and HeLa cells, respectively, indicating that PC-3 cells were more sensitive to the exogenous p53 transfection than HeLa cells. Meanwhile, G1 phase arrest was detected in PC-3 cells while a unique G2 phase arrest was identified in HeLa cells after p53 transfection. Through Western blotting, activity analysis of caspase-3, caspase-8 and caspase-9 and mitochondrial membrane potential measurement, the apoptosis induced by PEN-mediated p53 transfection was conducted in a mitochondria-dependent apoptosis pathway. These results demonstrated that PEN could successfully mediate the p53 gene delivery and up-regulate the cellular p53 expression level, triggering a significant p53-dependent anti-proliferative effect on tumor cells.
在本文中，通过迈克尔加成反应构建了N-异丙基丙烯酰胺修饰的聚乙烯亚胺（PEN），并将其用作载体以实现p53基因传递，使用HeLa（p53野生型）和PC-3细胞（p53缺失型）作为模型。在PEN介导的p53转染后，HeLa和PC3细胞中p53的表达水平在mRNA和蛋白质水平均上调。由于外源性p53的表达，通过MTT分析观察到细胞增殖受到抑制，这归因于凋亡的激活和细胞周期停滞。使用流式细胞术分析，在PEN介导的p53转染后，PC-3和HeLa细胞中的早期凋亡率分别检测到54.95%和27.06%，表明PC-3细胞对外源性p53转染比HeLa细胞更敏感。同时，在p53转染后，PC-3细胞中检测到G1期停滞，而HeLa细胞中鉴定出独特的G2期停滞。通过蛋白质免疫印迹法、caspase-3、caspase-8和caspase-9的活性分析以及线粒体膜电位测量，PEN介导的p53转染诱导的凋亡是在线粒体依赖性凋亡途径中进行的。这些结果表明，PEN可以成功介导p53基因传递并上调细胞p53表达水平，从而对肿瘤细胞引发显著的p53依赖性抗增殖作用。
