本文選題：AFT-SCF細(xì)胞系 + 干細(xì)胞因子　； 參考：《中國病理生理雜志》2016年04期

【摘要】：目的:構(gòu)建AFT024-SCF和HPC-Lhx2細(xì)胞系,并用HPC-Lhx2細(xì)胞系鑒定AFT024-SCF細(xì)胞系的生物學(xué)功能。方法:采用逆轉(zhuǎn)錄病毒感染法構(gòu)建干細(xì)胞因子(stem cell factor,SCF)依賴的永生化造血干/祖細(xì)胞(hematopoietic stem/progenitor cell,HPC)-Lhx2細(xì)胞系和小鼠胎肝基質(zhì)細(xì)胞系A(chǔ)FT024-SCF及其不含目的基因的對(duì)照組細(xì)胞系A(chǔ)FT024-GFP。采用real-time PCR法及Western blot法鑒定此AFT024-SCF細(xì)胞系中SCF的表達(dá)。ELISA法鑒定AFT024-SCF上清液中SCF的表達(dá)。收集AFT024-SCF及AFT024-GFP細(xì)胞培養(yǎng)上清液并以1∶10與IMDM基礎(chǔ)培養(yǎng)基混合備用。以AFT024-SCF組上清液為實(shí)驗(yàn)組,AFT024-GFP組上清液為內(nèi)源性陰性對(duì)照組,無任何添加的IMDM基礎(chǔ)培養(yǎng)基為外源性陰性對(duì)照組,添加重組SCF的IMDM培養(yǎng)基為陽性對(duì)照組,分別與HPCLhx2細(xì)胞系共培養(yǎng)72 h。MTT法檢測(cè)各組HPC-Lhx2細(xì)胞增殖活性,集落形成實(shí)驗(yàn)鑒定HPC-Lhx2細(xì)胞系擴(kuò)增后的細(xì)胞干性。結(jié)果:構(gòu)建的AFT024-SCF細(xì)胞系表達(dá)SCF;HPC-Lhx2細(xì)胞系體外培養(yǎng)72 h后,外源性及內(nèi)源性陰性對(duì)照組未能維持HPC-Lhx2細(xì)胞增殖;而陽性對(duì)照組及實(shí)驗(yàn)組均可促進(jìn)HPC-Lhx2細(xì)胞增殖;陽性對(duì)照組及實(shí)驗(yàn)組細(xì)胞均有集落形成單位,且差異無統(tǒng)計(jì)學(xué)意義,陰性對(duì)照組無集落形成單位。結(jié)論:成功構(gòu)建表達(dá)SCF的AFT024-SCF細(xì)胞系,其培養(yǎng)上清液能夠替代重組SCF用于HPC-Lhx2細(xì)胞系的體外擴(kuò)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.
【作者單位】： 中山大學(xué)附屬第三醫(yī)院血液內(nèi)科;安徽大學(xué);
【基金】：廣東省自然科學(xué)基金資助項(xiàng)目(No.S2013010016559;No.2014A030313138)
【分類號(hào)】：R329.2

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