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目的研究过继输注体外扩增同源调节性T细胞(Treg)对小鼠抗肿瘤免疫的影响,探索过继输注Treg治疗方法可能存在的风险。方法免疫磁珠分离法分离小鼠脾脏内CD4+CD25+Treg,流式细胞术测定其纯度后予以CD3/CD28单克隆抗体磁珠和大剂量IL-2(1 000 U/mL)刺激,进行2周2轮次扩增后收集Treg,混合淋巴细胞培养测定其体外抑制功能;后将1×107Treg静脉注射BALB/c小鼠,24 h后再注射B16F10肿瘤细胞,同时设置单独接种肿瘤细胞组,14 d后计数肺部移植瘤数目,测定外周血Treg比例。结果新鲜CD4+CD25+Treg纯度大于95%,平均96.3%±2.88%,体外扩增后纯度大于85%,平均87.73%±2.35%;与新鲜Treg相比,扩增后抑制功能未受损(P>0.05);给BALB/c小鼠注射1×106B16F10细胞后14 d肺部肿瘤结节数为(14±5)个,先注射1×107体外扩增Treg后再注射1×106B16F10细胞,肿瘤结节数增多为(73±9)个(P=0.007),与单独注射2×106B16F10细胞相当(86±8)个(P=0.230);给C57BL/6小鼠输注5×105B16F10细胞后结节数为(70±15)个,预先输入8×106体外扩增Treg后再注射5×105B16F10细胞,肺部肿瘤结节数明显增多,大于300个,与前者相比,差异有统计学意义(P<0.01),同时荷瘤小鼠外周血Foxp3+Treg比例上升更为显著(P<0.05)。结论过继输注体外扩增Treg诱导移植物免疫耐受的同时,可能抑制机体的抗肿瘤免疫,因此存在一定风险。
Objective To study the effect of adoptive infusion on proliferation of murine anti-tumor immunity by homologous regulatory T cells (Tregs) in vitro and to explore the possible risk of adoptive Treg treatment. Methods The CD4 + CD25 + Tregs were isolated from the spleens of mice by immunomagnetic bead separation. The purity of the CD4 + CD25 + Tregs was detected by flow cytometry. The cells were stimulated with CD3 / CD28 monoclonal antibody beads and high dose of IL-2 (1 000 U / mL) BALB / c mice were injected intravenously with 1 × 107 Treg at 24 h after injection of B16F10 tumor cells. Tumor cells were separately inoculated After 14 days, the number of lung xenografts was counted and the proportion of Treg in peripheral blood was measured. Results The purity of fresh CD4 + CD25 + Treg was greater than 95% with an average of 96.3% ± 2.88%. The purity of fresh CD4 + CD25 + Treg was over 85% with an average of 87.73% ± 2.35% after in vitro expansion. Compared with fresh Treg, P> 0.05). The number of lung tumor nodules in 14 days after inoculation of 1 × 106B16F10 cells into BALB / c mice was (14 ± 5). 1 × 106 T16 cells were injected with 1 × 107 in vitro before injection of 1 × 106B16F10 cells, The number of tumor nodules increased by 73 ± 9 (P = 0.007), which was 86 ± 8 compared with 2 × 106B16F10 cells alone (P = 0.230). C57BL / 6 mice were infused with 5 × 105B16F10 cells (70 ± 15) nodules, pre-input 8 × 106 in vitro expansion of Treg and then injected 5 × 105B16F10 cells, lung tumor nodules increased significantly, more than 300, compared with the former, the difference was statistically (P <0.01), while the proportion of Foxp3 + Treg in peripheral blood of tumor-bearing mice increased more significantly (P <0.05). Conclusion The adoptive infusion of Treg in vitro to induce graft immune tolerance may inhibit the body’s anti-tumor immunity, so there is a certain risk.