目的基因CXCL-1慢病毒表达系统的构建及在人正常肝细胞中的功能研究

目的：探讨含目的基因趋化因子生长调节基因1（CXCL-1）慢病毒表达系统的构建及CXCL-1对7702正常肝细胞生长、增殖、迁移生物学功能的影响。方法：构建含目的基因CXCL-1的慢病毒表达系统,建立携带GFP荧光标记并过度表达CXCL-1的7702正常肝细胞系,Western blot方法检测CXCL-1在转染细胞中的表达,细胞单克隆形成实验,MTT实验,Transwell实验检测7702细胞表达CXCL-1后生长、增殖和迁移能力的变化情况。结果：1成功构建目的基因CXCL-1慢病毒表达系统,病毒滴度检测结果为2.00E＋8TU/mL;2转染48h后经流式细胞仪检测,转染率达94.5%;3CXCL-1/7702组克隆形成率显著高于其余两对照组（P〈0.05）;4MTT比色法结果显示：CXCL-1/7702组分别与空载GFP/7702组及空白对照7702组相比较,生长速度明显较快（P〈0.05）;5Transwell实验显示：CXCL-1/7702组、GFP/7702组、7702组肝细胞系均无迁移能力。结论：成功构建含目的基因CXCL-1的慢病毒表达系统,CXCL-1对正常肝细胞具有明显的促细胞生长与增殖功能,但无促进正常肝细胞迁移的功能。

CONSTRUCTION OF THE LENTIVIRAL EXPRESSION SYSTEM OF CXCL-1 AND EFFECECTS OF CXCL-1 ON HEPATOCYTE

Objective：This study aimed to construct a lentiviral expression system of CXCL-1, and investigate the effect of expressed CXCL-1 on the cell growth, proliferation and transformation in the human normal liver cell line 7702. Methods： We constructed lentiviral expression system of CXCL-1, and then the normal liver cell line 7702 of carrying GFP fluorescence labeling and over-expression of CXCL-1 was built, the expression of CXCL-1 in the 7702 cell was determined by Western blot. Cell colony forming experiment and MTT were performed to evaluate the effects of expressed CXCL-1 on the cell growth and proliferation. Transwell experiment was used to analyze the effects on cell transformation. Results： （1）CXCL-1 gene lenti- viral expression system was constructed successfully, the virus titer was 2. 00E ＋ 8TU/mL. （2） After transfection of 48 h by flow cytometry, the efficiency of infection of the cells that express target gene was about 95%.（3）The clone formation and the number of clone formation rate were higher in group CXCL-1/ 7702 than in groups GFP/7702 and 7702 （ P 〈0. 05 for each）. （4）MTT assay results showed that the cell growth speed was faster in group CXCL-1/7702 than in groups GFP/7702 and 7702. （5）Transwell experiment showed that there was no migration ability of the groups CXCL-1/7702, GFP/7702 and 7702. Conclusion： CXCL-1 gene lentiviral expression system was constructed successfully. Chemokine CXCL-1 can obviously promote cell growth but not promote cell migration for normal liver cell line 7702.