AFT024-SCF细胞系的构建及其生物学功能鉴定

发布时间：2018-04-13 00:06

本文选题：AFT-SCF细胞系 + 干细胞因子　；参考：《中国病理生理杂志》2016年04期

【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。
[Abstract]:Aim: to construct AFT024-SCF and HPC-Lhx2 cell lines and to identify the biological functions of AFT024-SCF cell lines by HPC-Lhx2 cell line.Methods: an immortalized hematopoietic stem / progenitor cell line (HPC-Lhx2), a murine fetal liver stromal cell line (AFT024-SCF) and a control cell line AFT024-GFPwith no target gene were constructed by retrovirus infection.The expression of SCF in the AFT024-SCF cell line was identified by real-time PCR assay and Western blot method. The SCF expression in AFT024-SCF supernatant was identified by Elisa.The supernatants of AFT024-SCF and AFT024-GFP cells were collected and mixed with IMDM medium at 1:10.The supernatant of AFT024-SCF group was used as endogenous negative control group, no IMDM basal medium was exogenous negative control group, and the IMDM medium supplemented with recombinant SCF was positive control group.The proliferative activity of HPC-Lhx2 cells in each group was detected by 72 h.MTT method with HPCLhx2 cell line co-culture, and the dry property of HPC-Lhx2 cell line was evaluated by colony formation assay.Results: after 72 h culture of AFT024-SCF HPC-Lhx2 cell line, exogenous and endogenous negative control group could not maintain the proliferation of HPC-Lhx2 cells, while the positive control group and experimental group could promote the proliferation of HPC-Lhx2 cells.There were colony forming units in positive control group and experimental group, and there was no colony forming unit in negative control group.Conclusion: the AFT024-SCF cell line expressing SCF was successfully constructed and its supernatant could replace the recombinant SCF for the expansion of HPC-Lhx2 cell line in vitro.
【作者单位】：中山大学附属第三医院血液内科;安徽大学;
【基金】：广东省自然科学基金资助项目(No.S2013010016559;No.2014A030313138)
【分类号】：R329.2

【相似文献】