转基因JAK3体外长期扩增T淋巴祖细胞的实验研究

Long-term expansion of T cell progenitors by JAK3 gene transduced primitive hematopoietic cells in vitro

目的探讨激活JAK3信号传导通路是否能够体外长期扩增调控T淋巴祖细胞并评价其定向分化潜能的可行性。方法构建含有JAK3基因的逆转录病毒载体MG I-F2JAK3,内含有绿色荧光蛋白(GFP)和两个可以结合小分子二聚化合物AP20187的结合位点F36v。将该载体转入小鼠的骨髓造血干细胞中,在无血清培养基X-V ivo15培养体系中扩增,分为4组:空白对照组;AP20187组;干细胞因子(SCF)组;AP20187+SCF组。对扩增的细胞进行流式细胞仪检测免疫分型,定向分化、胸腺内注射等研究。结果AP20187+SCF组可以使转导的造血细胞获得长期大量对数级的扩增,扩增50 d后细胞可以达到1.2×1012倍±0.2×1012倍,所扩增的细胞为最早期的T淋巴祖细胞,表型为C-k ith iCD44+CD25-TN。该祖细胞群在SCF+IL-7+IL-3培养条件下,可定向分化为Thy1.2阳性的T淋巴细胞,并在小鼠的胸腺内可定向分化为GFP+CD3+和GFP+CD4+双阳性的成熟T淋巴细胞。结论AP20187联合SCF激活JAK3信号,可以大量扩增最早期的T淋巴祖细胞。该祖细胞具有定向分化为成熟T淋巴细胞的功能。该系统有助于研究T淋巴细胞的发生发育。

Objective To explore whether activation of the JAK3-signaling pathway can stimulate long-term expansion of the earliest T cell progenitors from transduced primitive hematopoietic cells and evaluate their potential ability of committed differentiation. Methods A retrovirus vector （RV） containing JAK3 gene, two binding sites for chemical inducers of dimerization （AP20187）, and green fluoresecnce protein （GFP）, MGI-F2JAK3, was constructed. The RV vector MGI-F2JAK3 was then tranedueed into murine bone marrow hematopoietie cells. The transduced murine bone hematopoietie marrow ceils were divided equally into four groups blank control group （No drug group ）, AP20187 group （added with AP20187 only）, SCF group （ added with stem cell factor only） , and AP20187 ＋ SCF group. Cytometry was used to detect the GFP marker and observe the survival of ceils. The murine bone hematopoietic marrow cells expanded for 50 days were divided into 2 groups： one group was washed to remove the eytokines to observe their survival, and Air20187 ＋ SCF was added into the culture fluid of other group. Cytometry and CELLQuest v3.1 software analysis were used to analyze the phenotype of the cells of AP20187 ＋ SCF after 50 days＇ expansion. The C-kit^hi CD44^＋ CD25^- TN cells after 50 days＇ expansion were further cultured under the condition of SCF ＋ ILT_IL3 for 5 days. The differentiation rate of Thyl. 2 positive cells was observed by eytometry. Five Ly5. 2 mice underwent radiation of the dose of 600 cGy, and then injected with the expanded cells into the thymus. Three weeks later the mice were killed and their thymus glands were taken out to prepare suspension of single cells to undergo cytometry to observe the proportions of GFP positive CD3 and CD4 mature T lymphocytes. Results One week later the cells of the No Drug group all died, the cells of the AP20187 group and SCF group died 2 - 4 weeks later, however, the cells of the AP20187 ＋ SCF continued to grow and expanded up to 1012-fold after 50 days＇ culture. The ceils of the AP20187 ＋ SCF group with the cytokines washed died 2 more weeks later, and those with the cytokines washed and added with AF20187 ＋ SCF continued to grow. The phenotype of the expanded proportion was identified as the earliest T cell progenitors expressing C-kit^hi CD44^＋CD25^-TN （triple negative）. These progenitors could differentiate into Thyl. 2 ＋ T ceils in the presence of SCF ＋ IL-7 ＋ IL-3 culture condition. 32% - 96% of the mice thymus cell were GFP positive, 5% ＋0. 8% of the thymus cells were GFP ＋ CD^3 double positive, and 11.0% - 2.1% of the T lymphocytes were GFP ＋ CD4^＋ double positive. Conclusion AP20187 combined with SCF mediating the activation of JAK3 signaling can dramatically expand the earliest T cell progenitors subpopulation, and acquires the capacity to induce the differentiation into T mature cells. This system may help understand the T cell biology and provide a fundamental basement for gene therapy to immunodeficiency disease in the future.