CAJ | 학술논문

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.
목적 구건WTX CDS구전장중조만병독표체재체,감염결직장암SW620세포,건립WTX은정고표체결직장암SW620세포주.방법 장WTX기인전장CDS구삽입GV287만병독질립재체,중조재체여포장재체공전염인배신293T세포,수집상청、농축득도표체WTX적중조만병독.만병독감염결직장암SW620세포,형광현미경초보관찰감염효솔,G418진일보사선득도은정감염세포주.QPCR급Western blotting검측WTX과표체효솔.매부분균설립상응대조.결과 재체편단측서결과시성공구건료WTX중조표체재체;포장병독적도검측괄중,제시WTX표체만병독포장성공;만병독감염결직장암SW620세포후, WTX기인mRNA이급단백표체수평명현증고,유통계학의의,제시SW620결직장암세포WTX고표체은정주구건성공.결론건립만병독감염방법적결직장암SW620세포WTX과표체은정세포주,위진일보연구WTX대결직장암발생、발전적작용궤제제공분자생물학공구.
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.