南方医科大学学报

南方医科大学学报 ›› 2015, Vol. 35 ›› Issue (08): 1122-.

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应用重组慢病毒表达载体建立抑癌基因WTX稳定高表达结直肠癌SW620细胞系

马文霞，贺莉，刘椿，张庆玲，丁彦青   

  • 出版日期:2015-08-20 发布日期:2015-08-20

Establishment of a colorectal cancer SW620 cell line stably over-expressing Wilm’s tumor
on X chromosome using a recombinant lentivirus vector

  • Online:2015-08-20 Published:2015-08-20

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摘要： 目的构建WTX CDS区全长重组慢病毒表达载体，感染结直肠癌SW620细胞，建立WTX稳定高表达结直肠癌SW620细
胞株。方法将WTX基因全长CDS区插入GV287慢病毒质粒载体，重组载体与包装载体共转染人胚肾293T细胞，收集上清、
浓缩得到表达WTX的重组慢病毒。慢病毒感染结直肠癌SW620细胞，荧光显微镜初步观察感染效率，G418进一步筛选得到
稳定感染细胞株。QPCR及Western blotting检测WTX过表达效率。每部分均设立相应对照。结果载体片段测序结果示成功
构建了WTX重组表达载体；包装病毒滴度检测适中，提示WTX表达慢病毒包装成功；慢病毒感染结直肠癌SW620 细胞后，
WTX基因mRNA以及蛋白表达水平明显增高，有统计学意义，提示SW620结直肠癌细胞WTX高表达稳定株构建成功。结论
建立慢病毒感染方法的结直肠癌SW620细胞WTX过表达稳定细胞株，为进一步研究WTX对结直肠癌发生、发展的作用机制
提供分子生物学工具。

Abstract: Objective To construct a recombinant lentivirus vector for Wilm’s tumor on X chromosome (WTX) gene and
establish a colorectal cancer SW620 cell line with stable WTX over-expression. Methods The full length coding region of WTX
gene was amplified with PCR, and the amplified fragment was cloned into the lentivirus vector GV387. The recombinant
lentivirus vector was transfected in 293T cells for packaging the virus, which was then transfected into colorectal cancer SW620
cells. The stably transfected cells were selected with G418, and the cellular expressions of WTX mRNA and protein were
detected using quantitative PCR and Western blotting. Results The recombinant plasmid was successfully constructed as
verified by sequence analysis. Quantitative PCR and Western blotting results showed that trasnfection with the recombinant
lentivirus significantly increased the expression levels of WTX in SW620 cells. Conclusion We successfully established a
colorectal cancer cell lines with stable over-expression of WTX, which provides an essential cell model for studying the role of
WTX in the tumorigenesis and progression of colorectal cancer.