中文摘要：

目的探讨并优化制备基因工程Th17细胞的方法，评估制备的工程Th17细胞是否具有同天然Th17细胞类似的表型和功能。方法包装含RORγt基因的重组慢病毒pXZ9-RORγt和空载体对照质粒pXZ9。C57BL6小鼠CD4^＋CD25^-nfffveT细胞体外活化36h后依据感染慢病毒颗粒的不同及是否添加TGF—β和IL-6，分为五组：pXZ9-RORγt组、TGF—β＋IL-6组（pXZ9＋TGF-β＋IL-6组）、联合组（pXZ9-RORγt＋TGF—β＋IL-6组）、空载体对照组（pXZ9组）及空白对照组。流式细胞术检测各组IL-17A阳性细胞比例，荧光定量PCR检测各组细胞IL-17A、IL-17F、IL-21和IFN-γ表达水平。通过小鼠实验性变应性脑脊髓炎（EAE）模型评估各组细胞是否具有加重EAE的功能。结果①pXZ9-RORγt组、TGF—B＋IL-6组和联合组均制备出工程Th17细胞，其中联合组IL-17A阳性率达（60．59±8．15）％，明显高于pXZ9-RORγt组[（40．25±5．46）％]和TGF—B＋IL-6组[（14．36±5．27）％]。②各组工程Th17细胞均表达IL-17A与IL-17F，而IL-21仅在给予TGF—β和IL-6时明显升高。③各组工程Th17细胞均可加重小鼠EAE。结论成功建立了制备高效工程Th17细胞的方法，其中过表达RORγt基因与联合TGF—β和IL-6不仅可提高Th17细胞的制备效率，而且制备的工程Th17细胞在表型和功能上与天然Th17细胞相类似。

英文摘要：

Objective To generate engineering Thl7 cells from mice CD4 ^＋ CD25^- nayve T cells, and to evaluate whether the phenotypes or functions of these engineering cells were similar to natural Thl7 cells. Methods Recombinant lentivirus carrying mouse RORγt （pXZ9-RORγt） and mock control pXZ9 were generated by co-transfected three-plasmids into 293FT packing cells. CD4 ^＋ CD25^ - naive T cells were purified from mice spleens by magnetic activated cell sorting, and stimulated by anti-CD3ε, anti-CD28 mAb plus IL-2. The stimulated cells were further infected by pXZ9-RORγt or pXZ9 virus with or without polarization by TGF-13 plus IL-6 and divided into five groups： pXZ9-RORγt （group A）, pXZ9 ＋ TGF-13 ＋ IL-6 （group B ）, pXZ9-RORγt ＋ TGF-13 ＋ IL-6 （ group C ）, pXZ9 （ group D ） and control （ group E）. Production efficiency of engineering Thl7 cells was referred as the percentage of IL-17A producing cells. Cytokine production pro- files of these cells were assayed by reahime RT-PCR and cells function was evaluated by susceptibility of mouse experimental autoimmune encephalomyelitis （EAE）. Results （!）High-title lentivirus was prepared and was succeeded to transduce CD4 ^＋ CD25 - naive T cells. Forced expression of RORγt（ group A） resulted in （40.25 -＋ 5.46）% CD4 ^＋ CD25- naive T cells converted into engineering Thl7 ceils and the convert effi- ciency increased to （60.59 ±8.15）% in addition of TGF-β and IL-6（group C）, or decresed to （ 14.36 ± 5.27）% when presence of TGF-β and IL-6 only（group B）. （2）IL-17A, IL-17F and IL-21 production of pXZ9-ROR＇yt infected cells combined with TGF-β and IL-6 were most similar to natural Thl7 cells while cells over expression of RORγt alone showed deficiency in IL-21 production. （3）Both pXZ9-RORγt infected cells, TGF-β and IL-6 polarized ceils and polarized of ROR3,t transduced cells could promote the susceptibility to mouse EAE in C57BL6 mice models. Conclusion High yield of engineering Thl7 cells w