本文關(guān)鍵詞： 人工抗原提呈細(xì)胞 CD 嵌合抗原受體修飾T細(xì)胞 增殖 殺傷　出處：《南方醫(yī)科大學(xué)學(xué)報(bào)》2017年05期 　論文類型：期刊論文

【摘要】：目的構(gòu)建CD19特異性人工抗原提呈細(xì)胞(aAPC)用于體外激活擴(kuò)增CD19嵌合抗原受體(CAR)修飾T細(xì)胞(CD19-CAR-T),并考察其殺傷效應(yīng)。方法通過(guò)慢病毒介導(dǎo)的方法制備以NIH3T3為細(xì)胞骨架、表達(dá)共刺激分子CD86和/或CD137L的CD19特異性a APC(NIH3T3-CD19/86、NIH3T3-CD19/86/137L)。采用照射的CD19特異性a APC與CD19-CAR-T細(xì)胞按一定比例混合培養(yǎng)激活擴(kuò)增CD19-CAR-T細(xì)胞,臺(tái)盼藍(lán)染色法檢測(cè)并繪制CD19-CAR-T細(xì)胞生長(zhǎng)曲線;流式細(xì)胞術(shù)檢測(cè)CD19-CAR-T細(xì)胞CAR表達(dá)變化及分化表型;生物發(fā)光細(xì)胞毒性法檢測(cè)擴(kuò)增的CD19-CAR-T細(xì)胞體外靶特異殺傷效應(yīng)。結(jié)果流式檢測(cè)結(jié)果顯示NIH3T3-CD19/86和NIH3T3-CD19/86/137L細(xì)胞表面分別高表達(dá)CD19、CD86和/或CD137L分子;NIH3T3-CD19/86和NIH3T3-CD19/86/137L細(xì)胞都能夠高效擴(kuò)增CD19-CAR-T細(xì)胞,NIH3T3-CD19/86/137L細(xì)胞具有更好的擴(kuò)增效應(yīng),其與CD19-CAR-T細(xì)胞混合培養(yǎng)14 d后,擴(kuò)增的T細(xì)胞數(shù)量明顯高于NIH3T3-CD19/86細(xì)胞組(P0.05);同時(shí),與刺激前相比NIH3T3-CD19/86和NIH3T3-CD19/86/137L細(xì)胞刺激擴(kuò)增的T細(xì)胞中CD19-CAR-T細(xì)胞比例顯著增加(P0.05);擴(kuò)增的CD19-CAR-T細(xì)胞具有靶特異殺傷效應(yīng),能夠特異性殺傷CD19陽(yáng)性靶細(xì)胞;流式檢測(cè)顯示NIH3T3-CD19/86/137L擴(kuò)增的CD19-CAR-T細(xì)胞含有約20%作用中心記憶性T細(xì)胞。結(jié)論成功制備CD19特異性a APC,其可在體外特異性擴(kuò)增功能性CD19-CAR-T細(xì)胞,為初步建立制備高質(zhì)量臨床級(jí)CD19-CART細(xì)胞的方法提供了技術(shù)支撐。
[Abstract]:Objective to construct CD19 specific artificial antigen presenting cell (APC) to activate and amplify CD19 chimeric antigen receptor (CAR) to modify T cell (CD19-CAR-T) in vitro. Methods the cytoskeleton of NIH3T3 was prepared by lentivirus-mediated method. CD19 specific a APC(NIH3T3-CD19/86 expressing costimulatory molecule CD86 and / or CD137L. NIH3T3-CD19 / 86 / 137L). CD19-CAR-T cells were activated by mixed culture of irradiated CD19 specific a APC and CD19-CAR-T cells in a certain proportion. Trypan blue staining was used to detect and draw the growth curve of CD19-CAR-T cells. The changes of CAR expression and differentiation phenotype of CD19-CAR-T cells were detected by flow cytometry. In vitro target specific cytotoxicity of amplified CD19-CAR-T cells was detected by bioluminescence cytotoxicity assay. Results NIH3T3-CD19/86 and NIH3T3-CD were detected by flow cytometry. The surface of 19 / 86 / 137L cells respectively expressed CD19. CD86 and / or CD137L molecules; Both NIH3T3-CD19/86 and NIH3T3-CD19/86/137L cells can amplify CD19-CAR-T cells efficiently. NIH3T3-CD19/86/137L cells had better amplification effect and were mixed with CD19-CAR-T cells for 14 days. The number of expanded T cells was significantly higher than that of NIH3T3-CD19/86 cells. At the same time. The percentage of CD19-CAR-T cells in T cells stimulated by NIH3T3-CD19/86 and NIH3T3-CD19/86/137L cells increased significantly compared with those before stimulation. P0.05; The amplified CD19-CAR-T cells have target specific killing effect and can specifically kill CD19 positive target cells. Flow cytometry showed that CD19-CAR-T cells amplified by NIH3T3-CD19/86/137L contained about 20% central memory T cells. Conclusion the specificity of CD19 was successfully prepared. A. APC. It can specifically amplify functional CD19-CAR-T cells in vitro and provide technical support for the preliminary preparation of high quality clinical grade CD19-CART cells.
【作者單位】： 解放軍總醫(yī)院腫瘤中心實(shí)驗(yàn)室;南開大學(xué)醫(yī)學(xué)院;第二軍醫(yī)大學(xué)腫瘤研究所;
【基金】：國(guó)家自然科學(xué)基金(81372528) 國(guó)家高技術(shù)研究發(fā)展計(jì)劃“863”計(jì)劃(2014AA020704)~~
【分類號(hào)】：R730.51
【正文快照】： 海200433Construction of specific artificial antigen-presenting cells for in vitro activation of CD19 chi-meric antigen receptor T cellsPENG Yaojun1,WU Qiyan1,LIU Hongyu2,ZHAO Jian1,3,WEI Huafeng11Cancer Center Key Laboratory,General Hospital of PLA,Beiji

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