[摘 要] 目的：探讨IL-22通过STAT3信号轴损害NK细胞功能并促进膀胱癌细胞耐药的机制。方法：常规培养T24细胞，用 梯度递增法构建耐药T24/顺铂（DDP）细胞。将细胞分为对照组（不处理）、DDP组、IL-22组、IL-22 + DDP组、IL-22 + anti-IL-22 组、IL-22 + DDP + Statti（c STAT3抑制剂）组。qPCR法检测各组T24细胞中IL-22、cyclin D1、Bcl-2 mRNA的表达，WB法检测各组 细胞中BAX、BCL2和p-STAT3的表达，CCK-8法检测各组细胞的增殖活性，流式细胞术检测各组细胞的凋亡，ELISA 检测各 组细胞上清液中乳酸脱氢酶（LDH）、TNF-α、IFN-γ、颗粒酶B（GzmB）和穿孔素（PRF）蛋白的水平。结果：T24/DDP细胞对DDP 敏感性降低（P < 0.05）；其耐药相关基因P-糖蛋白（P-gp）、肺耐药蛋白（LRP)、多药耐药相关蛋白1（MRP1）和IL-22及其受体表达 水平均明显升高（均P < 0.05），说明T24/DDP细胞构建成功。与对照组比较，DDP组T24细胞的增殖活力明显降低、凋亡率升高、 BAX蛋白表达升高、BCL2表达下降（均P < 0.05）；与DDP组比较，IL-22 + DDP组T24细胞的增殖活明显升高、凋亡率明显下降、 BAX蛋白表达降低、BCL2表达升高（均P < 0.05），表明IL-22通过调节BAX/Bcl-2的表达促进T24细胞对DDP耐药性。与对照 组比较，IL-22组T24 细胞总细胞和核中 p-STAT3表达水平均明显升高（均 P < 0.05）；与IL-22组比较，IL-22 + anti IL-22组T24 细胞中 p-STAT3水平明显降低（P < 0.05），说明IL-22激活T24细胞中STAT3的磷酸化过程，并促进其转核。与对照组比较，DDP 组T24与NK92细胞共培养上清液中LDH、TNF-α、IFN-γ、GzmB及PRF蛋白水平均明显升高（均P < 0.05），与DDP组比较，IL-22 + DDP 组共培养上清液中上述蛋白水平均明显降低（均 P < 0.05）；与对照组比较，IL-22 组共培养上清液中 LDH、TNF-α、IFN-γ、 GzmB及PRF蛋白水平明显降低（均P < 0.05）；与IL-22组比较，IL-22 + Stattic组共培养上清液中上述蛋白水平均明显升高（均 P < 0.05），说明IL-22可降低NK92细胞对T24细胞的毒性，STAT3抑制剂可逆转此作用。与DDP组比较，IL-22 + DDP组T24细 胞增殖活力明显升高（P < 0.05）；与 IL-22 + DDP 组比较，IL-22 + DDP + Stattic 组 T24 细胞增殖活力明显降低（P < 0.05）；与 DDP 组比较，IL-22 + DDP 组 T24 细胞的凋亡率明显升高（P < 0.05）；与IL-22 + DDP组比较，IL-22 + DDP + Stattic组T24细胞 的凋亡率明显升高（P < 0.05），说明 IL-22 调控 STAT3 影响 T24 细胞 DDP 耐药性及 NK 细胞免疫功能。结论： IL-22 通过激活 STAT3信号轴促进T24细胞的DDP耐药性，抑制NK细胞功能。

[Abstract] Objective: To investigate the mechanism by which IL-2 promotes bladder cancer cell resistance by impairing NK cell function via the STAT3 signalling axis. Methods: Bladder cancer T24 cells were routinely cultured, and cisplatin-resistant T24/DDP cells were established through stepwise dose-escalation method. Cells were divided into the following groups: control, DDP, IL-22, IL-22 + DDP, IL-22 + anti-IL-22, and IL-22 + DDP + Stattic (a STAT3 inhibitor). The mRNA expression of IL-22, cyclin D1, and BCL2 was detected using qRT-PCR. Protein expression of BAX, BCL2, and phosphorylated STAT3 (p-STAT3) was analyzed using WB. Cell proliferation and apoptosis were assessed using the CCK-8 assay and flow cytometry, respectively. The levels of lactate dehydrogenase (LDH), TNF-α, IFN-γ, granzyme B (GzmB), and perforin (PRF) in the supernatant were measured using enzyme-linked immunosorbent assay (ELISA). Results: T24/DDP cells exhibited significantly reduced sensitivity to DDP (P < 0.05), accompanied by markedly elevated expression levels of drug resistance-associated genes (P-glycoprotein[P-gp]，lung drug resistance protein[LRP],and multidrug resistance-associated protein 1[MRP1]), as well as IL-22 and its receptor (all P < 0.05), indicating successful establishment of DDP-resistant cells. Compared with the control group, the DDP group showed decreased proliferation, increased apoptosis, upregulated BAX protein expression, and downregulated Bcl-2 protein expression in T24 cells (all P < 0.05). Compared with the DDP group, the IL-22 + DDP group showed significantly increased proliferative activity, decreased apoptosis rate, downregulated BAX, and upregulated BCL expression (all P < 0.05), suggesting that IL-22 promotes DDP resistance in T24 cells by modulating BAX/BCL2 expression. Compared with the control group, IL-22 stimulation significantly increased total and nuclear p-STAT3 expression in T24 cells (all P < 0.05), and this increase was significantly attenuated by pre-treatment with an IL-22 neutralizing antibody (IL-22 + anti-IL-22 group) (P < 0.05), indicating that IL-22 activates STAT3 phosphorylation and promotes its nuclear translocation in T24 cells. In the T24-NK92 co-culture system, the levels of LDH, TNF-α, IFN-γ, GzmB, and PRF in the supernatant were significantly increased in the DDP group compared with the control group (all P < 0.05). Co-treatment with IL-22 and DDP significantly reduced the levels of these cytotoxicity-related factors compared to the DDP group (all P < 0.05). Furthermore, IL-22 treatment alone significantly decreased the levels of these factors compared to the control group (all P < 0.05), while the addition of the STAT3 inhibitor Stattic (IL-22 + Stattic group) reversed this suppression, leading to significant elevations in these factors (all P < 0.05). These findings indicate that IL-22 diminishes the cytotoxicity of NK92 cells against T24 cells, which can be reversed by STAT3 inhibition. Regarding chemoresistance, T24 cell proliferative activity was significantly higher in the IL-22 + DDP group than in the DDP group (P < 0.05). This enhancement was abolished by Stattic, as evidenced by significantly lower activity in the IL-22 + DDP + Stattic group compared to the IL-22 + DDP group (P < 0.05). Consistently, the apoptosis rate was significantly decreased in the IL-22 + DDP group compared with the DDP group (P < 0.05), and Stattic co-treatment significantly increased the apoptosis rate compared to the IL-22 + DDP group (P < 0.05). These findings indicate that IL-22 regulates both DDP resistance in T24 cells and NK cell-mediated immune function via the STAT3 pathway. Conclusion: IL-22 promotes DDP resistance in T24 bladder cancer cells and suppresses NK cell function via activating the STAT3 signaling axis.

[基金项目] 2022年度佛山市自筹经费类科技创新项目（2220001005778）