摘要
目的制备自身失活型慢病毒载体为基础的鼠基因工程调节性T细胞（Treg细胞），检测其表型变化，并观察其增殖能力及免疫抑制能力。方法构建含鼠Foxp3基因及内部核糖体进入位点序列（IRES）和绿色荧光蛋白基因（GFP）的双顺反子自身失活型慢病毒载体。采用脂质体转染法将慢病毒载体三质粒转染293T细胞，经共培养法感染免疫磁珠分离的小鼠CD4^＋CD25-T细胞，流式细胞术（FCM）检测基因转染效率及细胞Foxp3、CD25、GITR、CTLA-4的表达，CCK-8法、ELISA法检测其增殖、免疫抑制能力及细胞因子分泌的变化。结果成功构建了慢病毒表达质粒pXZ208-IRES—GFP、pXZ208·Foxp3-IRES—GFP，包装的重组慢病毒滴度达10^6IU／ml。以pXZ208-IRES—GFP为阴性对照组，共培养法感染CD4^＋CD25-γ细胞，实验组细胞Foxp3、CD25、CTLA-4、GITR表达升高。在抗CD3e单抗、抗原呈递细胞存在条件下，实验组细胞增殖能力及IL-2、IL-4、IL-10和IFN-γ的分泌均低于阴性对照组（P〈0．01）；且该细胞可显著抑制CI）4^＋CD25-T细胞的增殖。结论成功构建了自身失活型慢病毒载体介导的鼠基因工程Treg细胞；基因工程Treg细胞具有与CD4^＋ CD25^＋ Treg细胞相似的表型及低增殖性和免疫抑制性。
Objective To prepare the genetic engineering regulatory T cells （Treg） via the self-inactivating （SIN） lentiviral vectors carrying Foxp3 gene, and assay the phenotype and abilities of its proliferation and immunosuppression. Methods The bieistronic SIN lentiviral transfer plasmid containing Foxp3 gene and internal ribosomal entry site-green fluorescent protein gene （IRES-GFP） was constructed. Human embryonic kidney 293T ceils were co-transfected using liposome by lentiviral packing system, which included the packa- ging plasmid A NRF, the transfer plasmid and the envelope plasmid VSVG. The efficiency of gene transduction and the expressions of Foxp3, CD25, GITR, CTLA-4 of CD4^＋ CD25^-T ceils, which were isolated by magnetic beads from the spleen, and then co-cultured with 293T cells, were detected by flow cytometry （FCM）. The proliferative and suppressive capacities of transduced T cells were estimated by Cell Count Kit- 8 （CCK-8） and the cytokiue production was performed by ELISA. Results The lentiviral transfer plasmid pXZ208-Foxp3-IRES-GFP was successfully constructed, the virus titers were above 10^6 IU/ml in the supernatant. pXZ208-IRES-GFP was used as control group. After cocuhured, the CD4^＋ CD25^-T cells expressed significantly higher Foxp3, CD25, GITR and CTLA-4 in experimental group than in control group. Upon stimulation with anti-CD3e and APCs, the proliferative capacity of Foxp3-transduced T cells and the produc- tion of IL-2, IL-4, IL-10, IFN-γ were significantly lower than those in control group （P 〈 0.01 ） ; Foxp3- transduced T cells also significantly inhibited the proliferation of CD4^＋ CD25^- T cells. Conclusions The genetic engineering Treg mediated by SIN lentiviral vectors are successfully constructed and their phenotype and function are similar to natural CD4^＋CD25^＋ Treg.
出处
《中华血液学杂志》
CAS
CSCD
北大核心
2009年第8期528-532,共5页
Chinese Journal of Hematology
基金
国家自然科学基金（30770915）
江苏省普通高校研究生科研创新计划（CX07S-039Z）
关键词
慢病毒
转录因子FOXP3
T淋巴细胞
调节性
Lentiviral vector
Transcription factor Foxp3
T-lymphocytes ceils, regulatory
