摘要
目的建立成纤维生长因子19(FGF19)稳定过表达的人大肠癌细胞系,为研究FGF19在大肠癌发生发展中的作用奠定基础。方法将FGF19编码基因片段克隆到慢病毒载体中,测序鉴定后转染293T细胞,进行病毒包装及纯化并测定病毒滴度。慢病毒在感染复数为50 MOI的条件下,转染大肠癌SW480和SW620细胞。嘌呤霉素筛选获得稳定过表达FGF19的细胞系;用聚合酶链反应和蛋白质印迹法鉴定SW620和SW480稳转细胞株中FGF19的表达情况;用Transwell实验检测细胞迁移活性。结果成功构建慢病毒FGF19过表达慢病毒载体,病毒滴度为4×10^(8)TU·mL^(-1)。与空载对照组(NC组)相比,稳定过表达FGF19的SW480-OE、SW620-OE细胞的FGF19 mRNA水平分别增高18.57倍(P<0.01)和32.29倍(P<0.01);SW480-NC、SW480-OE、SW620-NC及SW620-OE组中,FGF19蛋白相对表达水平分别为1.00±0.27,3.85±1.51,1.00±0.25及2.74±0.65;与NC组相比,SW480-OE和SW620-OE的FGF19蛋白表达水平均明显升高,差异均有统计学意义(均P<0.05)。SW480-OE和SW620-OE细胞的迁移细胞数相对NC组平均分别上调4.84倍(P<0.01)和4.51倍(P<0.05)。结论构建并获得相应的慢病毒感染稳转过表达FGF19大肠癌细胞株及其对照细胞,初步发现FGF19可促大肠癌细胞的迁移活性。
Objective To establish human colorectal cancer cell lines with stable fibroblast growth factor 19(FGF19)-overexpression and to study the role of FGF19 in the development of colorectal cancer.Methods The FGF19 encoding fragment was cloned into a lentiviral vector and transfected into 293T cells for virus packaging,and purification and the virus titer was determined after identification by sequencing.The virus was transfected into SW480 and SW620 cells under the multiplicity of infection was 50 MOI.The puromycin screening yielded cell lines with stably FGF19 overexpression.The FGF19 mRNA and protein expression in SW480 and SW620 stable transfer cells were detected by polymerase chain reaction and Western blotting,respectively,and the Transwell assay deterred cell migration activity.Results The recombinant lentiviral vector was successfully constructed with a viral titer of 4×10^(8)TU·mL^(-1).Compared with NC groups,FGF19 mRNA expression in SW480-OE,SW620-OE cells increased 18.57-fold(P<0.01)and 32.29-fold(P<0.01),respectively.The relative expression of FGF19 protein in SW480-NC group,SW480-OE group,SW620-NC group,and SW620-OE group were1.00±0.27,3.85±1.51,1.00±0.25,2.74±0.65.Compared with the NC groups,the expression level of FGF19protein in the SW480-OE group and SW620-OE group were increased significantly.The differences of the factors were significant(all P<0.05).Compared with the NC group,the migration cell number of SW480-OE and SW620-OE cells was increased 4.84-fold(P<0.01)and 4.51-fold(P<0.05),respectively.Conclusion The stable cell lines of SW480-OE and SW620-OE and their control cells were established,and it was preliminarily found that FGF19 could promote the migration activity of colorectal cancer cells.
作者
王婷
王燕
彭朵
阮颖
姚运红
赵毅
WANG Ting;WANG Yan;PENG Duo;RUAN Ying;YAO Yun-hong;ZHAO Yi(Basic Medical School,Guangdong Medical University,Dongguan 523808,Guangdong Province,China)
出处
《中国临床药理学杂志》
CAS
CSCD
北大核心
2022年第23期2850-2854,共5页
The Chinese Journal of Clinical Pharmacology
基金
国家自然科学基金资助项目(81572566)
广东省自然科学基金资助项目(2016A030313674)
广东省科技计划基金资助项目(2016A020215224)。
关键词
成纤维生长因子19
大肠癌
慢病毒载体
细胞迁移
fibroblast growth factor 19
colorectal cancer
lentiviral vector
cell migration
