离心法增加HaCaT细胞的基因转染效率  

Centrifugation Increases the Gene Transfection Efficiency of HaCaT Cells

作　　者：徐云飞 孟莎莎[1] 周卫辉[1] 李红梅[2] Xu Yunfei;Meng Shasha;Zhou Weihui;Li Hongmei(Pediatric Research Institute, Children＇s Hospital of Chongqing Medical University, Ministry of Education Key Laboratory of Child Development and Disorders, Chongqing Key Laboratory of Translalional Medical Research in Cognitive Development and Learning and Memory Disorders, China International Science and Technology Cooperation Base of Child Development and Critical Disorders, Chongqing 400014, China;Department of Dermatology, Children＇s Hospital of Chongqing Medical University, Chongqing 400014, China)

机构地区：[1]重庆医科大学附属儿童医院儿科研究所儿童发育疾病研究教育部重点实验室认知发育与学习记忆障碍转化医学重庆市重点实验室儿童发育重大疾病国家国际科技合作基地 [2]重庆医科大学附属儿童医院皮肤科,重庆400014

出　　处：《中国细胞生物学学报》2018年第6期999-1007,共9页Chinese Journal of Cell Biology

基　　金：重庆医科大学附属儿童医院临床研究项目(批准号:lcyj2014-6)资助的课题~~

摘　　要：HaCaT细胞是自发性的人永生化表皮细胞株,常用于皮肤功能与疾病的相关研究,但常规脂质体法转染该细胞的效率极低。该研究主要探讨离心是否能够增加脂质体法转染HaCaT细胞的效率。以Lipofectamine~?2000为脂质体转染试剂,pEGFP-C1为质粒转染HaCaT细胞后分别使用常规法和离心法处理,利用荧光倒置显微镜和流式细胞仪测定转染效率;细胞增殖活力实验检测离心是否对细胞活力产生影响;荧光素酶报告基因、Western blot实验检测离心法实际效应对目的基因表达的影响。结果表明,离心法处理组所测得的转染效率均高于常规法处理组,差异有统计学意义。荧光素酶报告基因实验、Western blot实验则进一步证实了转染后离心的性价效应能增加目的基因的表达,具有较高的实用性。综上所述,常规转染步骤后离心能够增加HaCaT细胞的基因转染效率。HaCaT cells are spontaneous human immortalized epidermal cell lines that are commonly used in the study of skin function and disease but the efficiency of conventional liposome transfection of the cells is extremely low. This study is aimed to investigate whether centrifugation can increase the efficiency of liposomal transfection of HaCaT cells. Lipofectamine~？ 2000 was used as a liposomal transfection reagent, and p EGFP-C1 was used as a plasmid, and HaCaT cells were transfected by conventional method and centrifuged method respectively. Transfection efficiency was observed by inverted fluorescence microscope, quantitatively determined by flow cytometry. Cell proliferation activity was measured to determine whether centrifugation affect cell viability. The luciferase reporter assay and Western blot were carried out to detect the effect of centrifugation on the expressionof the target gene. The results showed that the transfection efficiency of centrifugation treatment was higher than that of the conventional treatment. The difference was statistically significant. Luciferase reporter gene assay and Western blot experiment further confirmed that the centrifugation after transfection could promote the expression of the target gene, which had high practicability. In summary, the transfection efficiency of HaCaT cells can be increased by centrifugation after conventional transfection procedures.

关 键 词：离心 转染效率 HACAT细胞 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]