摘要
目的:构建AFT024-SCF和HPC-Lhx2细胞系,并用HPC-Lhx2细胞系鉴定AFT024-SCF细胞系的生物学功能。方法:采用逆转录病毒感染法构建干细胞因子(stem cell factor,SCF)依赖的永生化造血干/祖细胞(hematopoietic stem/progenitor cell,HPC)-Lhx2细胞系和小鼠胎肝基质细胞系AFT024-SCF及其不含目的基因的对照组细胞系AFT024-GFP。采用real-time PCR法及Western blot法鉴定此AFT024-SCF细胞系中SCF的表达。ELISA法鉴定AFT024-SCF上清液中SCF的表达。收集AFT024-SCF及AFT024-GFP细胞培养上清液并以1∶10与IMDM基础培养基混合备用。以AFT024-SCF组上清液为实验组,AFT024-GFP组上清液为内源性阴性对照组,无任何添加的IMDM基础培养基为外源性阴性对照组,添加重组SCF的IMDM培养基为阳性对照组,分别与HPCLhx2细胞系共培养72 h。MTT法检测各组HPC-Lhx2细胞增殖活性,集落形成实验鉴定HPC-Lhx2细胞系扩增后的细胞干性。结果:构建的AFT024-SCF细胞系表达SCF;HPC-Lhx2细胞系体外培养72 h后,外源性及内源性阴性对照组未能维持HPC-Lhx2细胞增殖;而阳性对照组及实验组均可促进HPC-Lhx2细胞增殖;阳性对照组及实验组细胞均有集落形成单位,且差异无统计学意义,阴性对照组无集落形成单位。结论:成功构建表达SCF的AFT024-SCF细胞系,其培养上清液能够替代重组SCF用于HPC-Lhx2细胞系的体外扩增。
AIM： To construct AFT024-SCF cell line and HPC-Lhx2 cell line for confirming the biological function of AFT024-SCF. METHODS： The HPC-Lhx2 cell line,AFT024-SCF cell line and AFT024-GFP cell line were constructed by retro-viral infection. The expression of stem cell factor（ SCF） in AFT024-SCF cells was detected by realtime PCR and Western blot. SCF in the supernatant of AFT024-SCF was detected with ELISA. The supernatant of AFT024-SCF and AFT024-GFP were collected and then diluted（ 1∶10） with basic IMDM medium. So we made 4 culture medium：AFT024-SCF medium was used for experiment group,AFT024-GFP medium was used for endogenous negative control,IMDM basic medium was used for exogenous negative control,and IMDM basic medium with SCF was used for positive control. SCF-dependent HPC-Lhx2 cell line was cultured in these 4 different medium for 72 h. According to MTT method and colony forming experiment,the biological function of AFT024-SCF was confirmed by the proliferation ability of SCF-dependent HPC-Lhx2 cell line. RESULTS： SCF was highly expressed in AFT024-SCF cells. After cultured for 72 h,neither IMDM basic medium nor GFP-AFT024 medium support HPC-Lhx2 cell line proliferation. However,AFT024-SCF medium supported HPC-Lhx2 cell line expansion as well as the positive control medium. CONCLUSION： AFT024-SCF cells express SCF successfully and recombinant SCF can be replaced by the supernatant of AFT024-SCF culture medium for expanding HPC-Lhx2 cell line in vitro.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2016年第4期745-751,共7页
Chinese Journal of Pathophysiology
基金
广东省自然科学基金资助项目(No.S2013010016559
No.2014A030313138)
