探讨华蟾素调控肌球蛋白重链 9（MYH9）/泛素特异性蛋白酶7（USP7）/骨髓细胞瘤病毒癌基因（c-MYC）通路对急 性髓系白血病（AML）细胞免疫逃逸的影响。【方法】（1）体内实验：建立裸鼠异种移植瘤模型，评估华蟾素对AML细胞在体内 生长和免疫逃逸的影响。（2）体外实验：使用不同浓度的华蟾素处理人AML细胞株HL-60，细胞计数试剂盒8（CCK-8）法检测 细胞活力，Transwell实验检测细胞侵袭能力。将HL-60细胞与活化的CD8+ T细胞共培养，流式细胞术检测CD8+ T细胞表面 标志物 CD25的表达，酶联免疫吸附分析（ELISA）检测共培养上清液中细胞因子[白细胞介素 2（IL-2）和干扰素（IFN-γ）]的水 平，CytoTox96 非放射性细胞毒性分析评估CD8+ T细胞对HL-60细胞的毒性。Western Blot法检测MYH9、USP7和c-MYC的 蛋白表达，免疫共沉淀（Co-IP）法检测 MYH9、USP7 和泛素化之间的相互作用。转染 MYH9 过表质粒，验证华蟾素在 AML 中的作用机制。【结果】 华蟾素抑制裸鼠移植瘤生长，增强CD8+ T细胞抗肿瘤的能力。华蟾素浓度依赖性地抑制HL-60细胞 活力、侵袭。华蟾素处理后上调 CD8+ T细胞表面标志物 CD25的表达，同时还上调 IL-2和 IFN-γ水平。华蟾素增强 CD8+ T 细胞对 HL-60细胞的毒性。华蟾素抑制 HL-60细胞 MYH9、USP7和 c-MYC 的蛋白表达，MYH9通过募集 USP7促进 c-MYC 去泛素化，华蟾素抑制MYH9介导的c-MYC去泛素化。【结论】 华蟾素可通过抑制MYH9的表达进而减少去泛素化酶USP7对 c-MYC的募集，促进c-MYC泛素化降解，从而抑制AML细胞免疫逃逸。

To investigate the effect of cinobufacini on immune escape of acute myeloid leukemia （AML） by regulating myosin heavy chain 9（MYH9）/ubiquitin-specific protease 7（USP7）/cellular-myelocytomatosis viral oncogene（c-MYC） pathway. Methods （1）In vivo experiment： a nude mouse xenograft tumor model was established to evaluate the effect of cinobufotalin on the growth and immune escape of AML cells in vivo.（2）In vitro experiments：human AML cell line HL-60 was treated with different concentrations of cinobufacini，cell viability was detected by cell counting kit 8（CCK-8），and HL-60 cell invasion was detected by Transwell assay. HL-60 cells were co-cultured with activated CD8+ T cells，the expression of CD25，the surface marker of CD8+ T cells， was detected by flow cytometry，the levels of cytokines [interleukin-2（IL-2） and interferon（IFN-γ）] in the coculture supernatant were detected by enzyme-linked immunosorbent assay（ELISA）. CytoTox96 non-radioactive cytotoxicity assay was used to evaluate the cytotoxicity of CD8+ T cells to HL-60 cells. The protein expressions of MYH9，USP7 and c-MYC in HL-60 cells were detected by Western Blot. The interaction between MYH9，USP7 and ubiquitination was detected by co-immunoprecipitation（Co-IP） assay. The MYH9 overexpression plasmid was tranfected to verify the mechanism of cinobufacini in AML. Results Cinobufacini treatment inhibited xerograft tumor growth in nude mice and enhanced the anti-tumor ability of CD8+ T cells. Cinobufacini treatment inhibited HL-60 cell viability and invasion in a concentration-dependent manner. Cinobufacini treatment up-regulated the expression of CD25， a surface marker of CD8+ T cells， and also up-regulated the levels of IL-2 and IFN- γ. Cinobufotalin enhanced the toxicity of CD8+ T cells to HL-60 cells. Cinobufacini inhibits the protein expressions of MYH9， USP7 and c-MYC in HL-60 cells. MYH9 promotes c-MYC deubiquitination by recruiting USP7， but cinobufacini inhibits MYH9-mediated c-MYC deubiquitination. Conclusion Cinobufacini can reduce the recruitment of c-MYC by deubiquitinating enzyme USP7 by inhibiting the expression of MYH9，and promote the ubiquitination and degradation of c-MYC，thereby inhibiting the immune escape of AML cells.

R285.5

湖南省自然科学基金资助项目（编号：2021JJ30525）；湖南省临床医疗技术创新引导项目（编号：2022SK51405）