目的: 研究抗衰老蛋白Klotho 对荷宫颈癌小鼠体内调节性T细胞（Treg 细胞）和辅助性T细胞17（Th7）细胞介导宫颈癌细胞免疫逃逸的影响及其作用机制。方法: 建立宫颈癌U14 细胞移植瘤小鼠模型，并设Control 组（正常小鼠组）、Model 组（荷宫颈癌小鼠模型组）、Klotho 处理组（荷宫颈癌小鼠经Klotho 蛋白处理组，200 ng/d)。分别在处理7、14 d 时称取各组小鼠体内宫颈癌移植瘤质量，以流式细胞术检测各组小鼠脾淋巴细胞功能、Treg和Th7细胞比例变化，qPCR检测各组小鼠Treg和Th7细胞关键转录因子Foxp3、RORγt的表达情况，ELISA方法检测各组小鼠脾淋巴细胞培养液中IL-17、IL-6、IL-10、TGF-β、IL-23 等因子的含量变化，WB检测各组小鼠脾淋巴细胞中Klotho、TGF-β1、Foxp3、RORγt 蛋白表达的变化。结果: 14 d 时，Klotho 组宫颈癌荷瘤小鼠肿瘤抑瘤率显著高于Model 组[(52.16±8.25)% vs (23.33±6.29)%，P<0.05]。Model 组与Control 组相比，荷瘤小鼠脾淋巴细胞中Treg、Th7 细胞比例均显著升高（均P<0.05），总T淋巴细胞（CD3+）、辅助/诱导T淋巴细胞（CD3+CD4+）比例及免疫指数（CD3+CD4+/CD3+CD8+）显著下降（均P<0.05），Foxp3、RORγt 基因mRNA表达显著增加（均P<0.05），IL-17、IL-6、IL-10、TGF-β、IL-23 等因子分泌显著增加（均P<0.05），Klotho 蛋白水平明显下降（P<0.05），TGF-β1、Foxp3、RORγt 蛋白表达均明显升高（均P<0.05）；而Klotho 处理组与Model 组相比，上述指标均出现了相反的变化（均P<0.05），但与Control 组无显著差异（均P>0.05）。结论: Klotho 蛋白可能通过调控TGF-β1/Foxp3/RORγt信号通路抑制宫颈癌荷瘤小鼠体内Treg和Th7 细胞介导的免疫逃逸，从而发挥抗肿瘤作用。

Objective: To study the effect of anti-aging Klotho protein on immune escape mediated by regulatory T cells (Treg)/helper T cells 17 (TH17) in mice bearing cervical cancer and its mechanism. Methods: The model of cervical cancer-bearing mice were established,and the control group (normal mice), model group (cervical cancer-bearing mice model), and Klotho treatment group (cervical cancer-bearing mice treated with Klotho protein, 200 ng/d) were set up. The weight of cervical cancer tumors in mice of each group was weighed at 7 and 14 days after treatment respectively, PBMCs were separated at the same time. Flow cytometry was used to detect the changes of T lymphocyte function and the proportion of Treg and TH17 cells in mice. qPCR was used to detect the expressions of Foxp3 and RORγt, the key transcription factors of Treg/TH17 cells, in PBMCs of mice in each group. The changes of IL-17, IL-6, IL-10, TGF-β and IL-23 in PBMCs were detected by ELISA. The protein expressions of Klotho, TGF-β, Foxp3 and RORγt in PBMCs of mice were detected by WB assay. Results: On the 14th day, the tumor inhibition rate of the cervical cancer-bearing mice in the Klotho group was significantly higher than that in the Model group [(52.16±8.25)% vs (23.33±6.29)% the model group to be supplemented, P<0.05). Compared with the Control group, the ratios of Treg and TH17 cells in the lymphocytes of the tumor-bearing mice significantly increased (all P<0.05), the ratios of total T lymphocytes (CD3+ ), auxiliary / induced T lymphocytes (CD3+CD4+ ) and immune index (CD3+CD4+/CD3+CD8+ cells) decreased significantly (all P<0.05); in addition, the mRNA expressions of Foxp3 and RORγt genes, cytokines of IL-17, IL-6, IL-10, TGF-β and IL-23, as well as protein expressions of TGF-β1, Foxp3 and RORγt increased significantly (all P<0.05), while the level of Klotho protein significantly decreased in Model group (P<0.05). Compared with the Model group, the above indicators showed opposite changes in Klotho group (P<0.05), but there was no significant difference with the Control group (all P>0.05). Conclusion: Klotho protein may inhibit Treg/TH17 cell-mediated immune evasion in cervical cancer-bearing mice by inhibiting TGF-β1/Foxp3/RORγt signaling pathway and exert anti-tumor effect.

河北省医学科学重点课题计划资助项目（No.20171182)