SENEX基因介导急性髓细胞白血病免疫逃逸的机制研究

Study on the Mechanism of SENEX Gene-Mediated Immune Escape in Acute Myeloid Leukemia

摘要: 目的：SENEX基因介导急性髓细胞白血病免疫逃逸的机制研究。方法：(1) 留取实验组患儿及正常健康对照儿童的血清，采用密度梯度离心法可得到外周血单个核细胞(Peripheral blood mononuclear cell, PBMC)。将CD4⁺ CD25⁺ CD127low界定为Treg细胞的表型标志，分别采用流式细胞术(Flow Cytometry, FC)、实时定量PCR (QRT-PCR)和酶联免疫吸附测定(ELISA)，测定相关样本中Treg细胞的占比、FoxP3 mRNA的表达量，以及血清中TNF-α、IL-10、IL-17、TGF-β1等细胞因子的浓度。(2) 采集3名健康儿童对照和4名初诊AML患儿的外周血，各取10 ml；分离并保留血清，经密度梯度离心获得外周血单个核细胞。利用流式细胞术分选并纯化CD4⁺ CD25⁺ Treg细胞与CD4⁺ CD25⁻效应T细胞(Teffs)，并且在体外进行短时间的原代培养，24小时后评估自发性凋亡；并以不同浓度H₂O₂诱导氧化应激损伤，通过流式分析Annexin V (+)凋亡细胞的比例。(3) 采用RNA干扰策略特异性下调Treg/Teff细胞中SENEX基因的表达，在体外建立SENEX-siRNA转染的Treg/Teff细胞模型。运用QRT-PCR检测Treg/Teff细胞内SENEX及促凋亡相关基因P53、P16、P21、Caspase-3的mRNA表达水平，并用流式细胞术统计Annexin V (+)凋亡细胞的比例。结果：(1) 初诊儿童AML外周血Treg细胞比例显著增高。同时，AML患儿外周血FoxP3基因mRNA表达显著高于儿童健康对照(p < 0.05)。与健康儿童对照组相比，AML患儿血清中IL-10 (11.17 ± 1.08 pg/ml)、IL-17 (21.78 ± 3.69 pg/ml)、TGF-β1 (12.51 ± 1.02 ng/ml)及TNF-α (21.35 ± 4.16 pg/ml)的水平均显著升高；而其血清IL-2浓度(165.29 ± 26.45 pg/ml)较对照组(409.24 ± 90.81 pg/ml)明显降低，差异有统计学意义(p < 0.05)。(2) 初诊AML患儿外周血中Treg细胞的自发性凋亡率明显低于Teff细胞；H₂O₂诱导的Treg/Teff细胞凋亡呈剂量依赖关系，在相同剂量H₂O₂作用下，Teff细胞的诱导凋亡率始终高于Treg细胞，差异具有统计学意义。(3) 以100 μM H₂O₂处理2小时，可明显提高AML患儿Treg与Teff细胞的凋亡比例，分别达到11.2% ± 2.6%和13.1% ± 4.3%。在体外建立SENEX基因SiRNA转染Treg/Teff细胞模型后发现，与单纯H₂O₂处理2 h的Treg细胞比较，转染后加用H₂O₂处理2 h的Treg细胞凋亡相关基因P53、P16、P21和Caspase-3 mRNA表达均明显升高(2-ΔΔCT分别为6705.73 ± 1124.07、253.08 ± 16.01、154.58 ± 35.12和5135.79 ± 985.47；p < 0.05)。此外，在相同处理条件下，干扰SENEX基因表达的转染组可显著提高Treg细胞的凋亡率(21.5 ± 3.4%)；在Teff细胞(13.9 ± 3.1%)中则未观察到该抗凋亡效应。这说明，H₂O₂诱导下SENEX基因能够抵抗Treg细胞凋亡，但对Teff细胞凋亡无抵抗作用。结论：在AML发病进程中，SENEX基因通过P16INK4A/Rb途径诱导细胞周期停滞，使AML患者Treg细胞获得抗凋亡能力，促使其外周Treg累积，从而促进了AML细胞的免疫逃逸。

Abstract: Objective: To investigate the mechanism by which the SENEX gene mediates immune escape in acute myeloid leukemia (AML). Methods: (1) Serum was collected from children in the experimental group and healthy control children. Peripheral blood mononuclear cells (PBMCs) were obtained by density gradient centrifugation. CD4⁺ CD25⁺ CD127low was defined as the phenotypic marker of Treg cells. The proportion of Treg cells, the expression level of FoxP3 mRNA, and the concentration of cytokines such as TNF-α, IL-10, IL-17, and TGF-β1 in the relevant samples were measured by flow cytometry (FC), quantitative real-time PCR (QRT-PCR), and enzyme-linked immunosorbent assay (ELISA). (2) Peripheral blood specimens (10 ml per sample) were obtained from 3 healthy children and 4 children newly diagnosed with AML. Serum was separated and retained, and peripheral blood mononuclear cells were obtained by density gradient centrifugation. PBMCs were isolated, and CD4⁺ CD25⁺ Tregs and CD4⁺ CD25⁻ Teffs were purified and sorted by flow cytometry. Furthermore, the cells were cultured in vitro for a short period of time, and spontaneous apoptosis was assessed after 24 hours. Oxidative stress damage was induced by different concentrations of H₂O₂, and the proportion of Annexin V (+) apoptotic cells was analyzed by flow cytometry. (3) RNA interference was employed to selectively knock down SENEX expression in Treg/Teff cells, thereby establishing an in vitro SENEX-siRNA transfection model. mRNA transcripts of SENEX and proapoptotic genes (P53, P16, P21, Caspase-3) levels in Treg and Teff cell groups were determined by QRT-PCR; apoptosis rate (Annexin V-positive cells%) was detected by flow cytometry. Results: (1) Peripheral blood Treg frequency was markedly higher in children with newly diagnosed AML. Simultaneously, FoxP3 gene mRNA expression in AML children was significantly higher than in healthy controls (p < 0.05). Compared to the healthy control group, serum concentrations of IL-10 (11.17 ± 1.08 pg/ml), IL-17 (21.78 ± 3.69 pg/ml), TGF-β1 (12.51 ± 1.02 ng/ml), and TNF-α (21.35 ± 4.16 pg/ml) were significantly elevated in AML children, while the serum IL-2 concentration (165.29 ± 26.45 pg/ml) was significantly lower than in healthy controls (409.24 ± 90.81 pg/ml, p < 0.05). (2) The spontaneous apoptosis rate of Tregs from newly diagnosed AML children was significantly lower than that of Teff cells. H₂O₂-induced apoptosis in Treg/Teff cells showed a dose-response relationship. At the same H₂O₂ concentration, the induced apoptosis rate was consistently higher in Teff cells than in Tregs, with a statistically significant difference. (3) Treatment with 100 μM H₂O₂ for 2 hours significantly increased the apoptosis rate in both Tregs (11.2% ± 2.6%) and Teff cells (13.1% ± 4.3%) from AML children. The SENEX gene siRNA transfection model in Treg/Teff cells was successfully established in vitro. Compared with Treg cells treated with H₂O₂ alone for 2 h, the expression of apoptosis-related genes P53, P16, P21 and Caspase-3 mRNA in Treg cells after transfection and subsequent H₂O₂ treatment for 2 h was significantly increased (2-ΔΔCT values were 6705.73 ± 1124.07, 253.08 ± 16.01, 154.58 ± 35.12 and 5135.79 ± 985.47, respectively; p < 0.05). Under the same treatment conditions, SENEX-siRNA transfection significantly increased the apoptosis rate of Tregs (21.5 ± 3.4%). However, no SENEX-mediated anti-apoptotic effect was observed in Teff cells (apoptosis rate 13.9 ± 3.1%). These results indicate that the SENEX gene can antagonize H₂O₂-induced apoptosis in Tregs but not in Teff cells. Conclusion: In the pathogenesis of AML, the SENEX gene induces cell cycle arrest through the P16INK4A/Rb pathway, enabling AML patient Treg cells to acquire anti-apoptotic capabilities, promoting the accumulation of peripheral Tregs, and thus facilitating the immune escape of AML cells.