结核杆菌抗原85B抑制自噬促进霍奇金淋巴瘤细胞凋亡机制研究

Mechanism by which mycobacterial antigen 85B inhibits autophagy and promotes apoptosis in Hodgkin lymphoma cells

摘要:

目的 探讨结核杆菌抗原85B（mycobacterial antigen 85B, Ag85B）抑制自噬促进霍奇金淋巴瘤（Hodgkin lymphoma, HL）细胞凋亡及机制。方法 回顾性收集新疆医科大学第一附属医院收治的80例HL及同期30例淋巴结反应性增生患儿（对照组）临床资料及病理组织切片。采用免疫组化分析微管相关蛋白1轻链3（microtubule-associated protein 1 light chain 3, LC3）、自噬降解底物蛋白1（sequestosome 1, P62/SQSTM1）、Beclin-1在HL及对照组患儿病理组织中的表达。将人霍奇金淋巴瘤细胞（HDLM-2）分为HDLM-2组、HDLM-2+结核杆菌抗原85B（mycobacterial antigen 85B, Ag85B）组（Ag85B分别为0.5、1、2、4 μg/mL）。采用CCK8法检测HDLM-2细胞增殖；实时荧光定量聚合酶链式反应法检测LC3、P62、Beclin-1、磷脂酰肌醇3-激酶（phosphatidylinositol-3-kinase, PI3K）、蛋白激酶B（protein kinase B, Akt）、哺乳动物雷帕霉素靶蛋白（mammalian target of rapamycin, mTOR）mRNA的表达；凋亡试剂盒检测细胞凋亡。结果 HL组LC3、Beclin-1阳性表达高于对照组（P<0.05），P62阳性表达低于对照组（P<0.05）；Ⅲ~Ⅳ期较Ⅰ~Ⅱ期LC3、Beclin-1阳性表达增高，P62阳性表达降低（P<0.05）。细胞实验结果显示，HDLM-2+Ag85B组与HDLM-2组比较，细胞增殖受抑，LC3和Beclin-1的mRNA表达减低，P62、PI3K、Akt和mTOR的mRNA表达增高，细胞凋亡增加，且在Ag85B为2 μg/mL时，Ag85B干预HDLM-2细胞24 h作用最强，差异有统计学意义（P<0.05）。结论 自噬在HL患儿中增强，且随疾病的分期增加而增强；Ag85B可抑制HL肿瘤细胞增殖及自噬，促进细胞凋亡，其机制可能与促进PI3K/Akt/mTOR通路相关。

Abstract:

Objective To investigate the mechanism by which mycobacterial antigen 85B (Ag85B) inhibits autophagy and promotes apoptosis in Hodgkin lymphoma (HL) cells.Methods The clinical data and pathological tissue slides were retrospectively collected from 80 HL children and 30 children with reactive lymphadenopathy (control group) treated at the First Affiliated Hospital of Xinjiang Medical University. Immunohistochemical analysis was performed to assess the expression of microtubule-associated protein 1 light chain 3 (LC3), sequestosome 1 (P62/SQSTM1), and Beclin-1 in the pathological tissues of HL and control groups. Human Hodgkin lymphoma cells (HDLM-2) were divided into the HDLM-2 group and the HDLM-2+Ag85B groups (with Ag85B concentrations of 0.5, 1, 2, and 4 μg/mL). The CCK8 method was used to measure HDLM-2 cell proliferation; qRT-PCR was employed to detect the expression of LC3, P62, Beclin-1, Akt, and mTOR mRNA in cells. An apoptosis kit was used to detect cell apoptosis.Results The positive expression of LC3 and Beclin-1 in the HL group were higher than those in the control group (P<0.05), while the positive expression of P62 was lower than that in the control group (P<0.05). In stages III-IV compared to stages I-II, the positive expression of LC3 and Beclin-1 increased, while the positive expression of P62 decreased (P<0.05). Cell experiment results showed that the HDLM-2+Ag85B group had suppressed cell proliferation compared to the HDLM-2 group, with decreased mRNA expression of LC3 and Beclin-1, and increased mRNA expression of P62, PI3K, Akt, and mTOR, leading to increased cell apoptosis. Notably, when Ag85B was at a concentration of 2 μg/mL, it had the strongest effect on HDLM-2 cells after 24 hours (P<0.05).Conclusions Autophagy is enhanced in children with HL and increases with disease stage. Ag85B can inhibit the proliferation and autophagy of HL tumor cells and promote apoptosis, possibly related to the activation of the PI3K/Akt/mTOR pathway.