PEI-壳聚糖在结肠癌细胞CD133^+梯度表达的转染研究

Transfection Efficiency Using PEI-CP Complex for CD133^+ Differently Expressed by Colon Cancer Lines

目的:以PEI-壳聚糖(CP)为载体材料,考察CD133^+呈梯度表达的4种结肠癌细胞上的转染效率,将特异性结合CD133的TR多肽修饰CP材料,探讨材料与转染的关系。方法:根据CD133^+含量将细胞分为高表达组和低表达组,合成法制备CP材料,激光粒度仪测定纳米粒粒径、电位,CCK8实验检测细胞毒性,用质粒p GL3和EGFP考察CP材料的转染效果,Western印记检测CD133^+蛋白的表达。结果:CD133^+高表达组SW480、HCT116的CD133^+含量为96.97%、92.96%,低表达组CaCO_2、SW620的CD133^+含量为2.00%、0.56%;CCK8结果得出,CP材料按质量比N/P=5时,生物安全性较高;质粒p GL3转染高表达组,其CD133^+RLU值低于低表达组;质粒EGFP转染高表达组,绿色荧光弱于低表达组,Western blot检测接TR肽的CP材料CD133^+表达高于CP材料。结论:CP材料在低表达CD133^+组转染效率高,且接TR肽的CP材料针对CD133^+有可能介导材料进入细胞。

Objective：To investigate transfect efficiency of CP/DNA at CSC marker CD133^＋ which is shown to be differently expressed in colon cancer cell lines,and to discuss the relationship between transfect and TR peptide,which is capable to specifically bind to CD133 by targeting CD133^＋ colon cancer cells.Methods：The expression of CD133^＋ in four cell lines were divided into 2 groups：the positive colon cell lines in SW480,HCT116,and the negative in CaCO2,SW620.Considering of PEI and Chitosan,they were linked together and composition,particle size,as well as the zeta potential of CP complex was measured.CCK-8 assay was also used to evaluate their toxicity.Using Chitosan-linked-PEI as the vector,PGL3 and EGFP plasmid transfect into four colon cancer cells after 48 h incubation,and the CD133-related protein expressions were assessed by Western blotting analysis.Results：The CSC marker expression of CD133^＋ were significantly different.CD133^＋ was positive in more than 90% of colon cells,SW480 in 96.97%,HCT116 in 92.96%,however,low expression of CaCO2,SW620 was found in 2.00%,0.56%.When the N/P of PEI/Chitosan/DNA was 5∶ 4∶ 1,CP complex shows a high transfect efficiency and low toxicity.And when the positive expression of CD133^＋ was added,the transfect efficiency was decreased.The result of Western blotting analysis showed that the ratios of CD133 in positive group were higher than those in negative group.Conclusion：The transfect efficiency of CP vector can increase the transfect efficient in low expression of CD133^＋ .TR peptide-CD133 may facilitate CP vector into cells.