PEI-壳聚糖在结肠癌细胞CD133<sup>+</sup>梯度表达的转染研究

doi:10.13523/j.cb.20160605

中国生物工程杂志

2016, Vol. 36

Issue (6): 32-38 DOI: 10.13523/j.cb.20160605

研究报告

PEI-壳聚糖在结肠癌细胞CD133⁺梯度表达的转染研究

代爽, 赵青青, 邱峰

重庆医科大学附属第一医院重庆 400016

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

DAI Shuang, ZHAO Qing-qing, QIU Feng

Department of Pharmaceutics, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China

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摘要：

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pGL3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CaCO₂、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pGL3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。

关键词： 非病毒载体; 转染; 结直肠癌; CD133⁺

Abstract:

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 48h 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.

Key words: Non-viral vector Transfection Colon cancer CD133⁺

收稿日期: 2016-02-17 出版日期: 2016-06-25

ZTFLH:

Q819

基金资助:

国家青年自然科学基金(81202771)资助项目

通讯作者: 邱峰 E-mail: qiufeng.cn@outlook.com

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引用本文:

代爽, 赵青青, 邱峰. PEI-壳聚糖在结肠癌细胞CD133⁺梯度表达的转染研究[J]. 中国生物工程杂志, 2016, 36(6): 32-38.

DAI Shuang, ZHAO Qing-qing, QIU Feng. Transfection Efficiency Using PEI-CP Complex for CD133⁺ Differently Expressed by Colon Cancer Lines. China Biotechnology, 2016, 36(6): 32-38.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20160605 或 https://manu60.magtech.com.cn/biotech/CN/Y2016/V36/I6/32

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