缺氧诱导肿瘤细胞BCL-2表达促进LPPCN样细胞核凝集的形成

Hypoxia induces expression of BCL-2 and formation of nuclear condensation as LPPCN

目的 模拟缺氧微环境分析缺氧条件下BCL-2表达及其对核凝集形成的影响,探讨线形程序性坏死（linearlypatterned programmed cell necrosis,LPPCN）样细胞核凝集形成的分子机制。方法 利用CoCl2缺氧模型模拟肿瘤细胞缺氧微环境,以B16黑色素瘤细胞系作为研究对象观察核凝集出现时肿瘤细胞的变化;利用伤口愈合实验、SRB实验评价缺氧对肿瘤细胞迁移、增殖的影响;应用Western blot和RT-PCR检测缺氧对凋亡相关分子BCL-2蛋白和RNA表达的影响;利用免疫荧光技术观察缺氧对BCL-2表达定位的影响。结果 缺氧36~48h后,采用Hochest33342活体细胞染色,荧光显微镜下可见30%以上胞核呈高度凝集状态,表现为单个凝集体或多个连接的小凝集体,未见明显的凋亡;缺氧可以明显抑制肿瘤细胞的增殖和迁移能力;在缺氧条件下,BCL-2表现出随时间变化的特征性表达谱,与肿瘤细胞核凝集出现的时间一致。结论 缺氧可诱导肿瘤细胞核凝集的形成,呈现与LPPCN细胞同样的特点;缺氧条件下存活的肿瘤细胞表现出更强的运动潜能;缺氧条件下抗凋亡蛋白BCL-2表达高峰的形成可能与核凝集有关。

Purpose Hypoxic microenvironment was mimicked to investigate expression of BCL-2 and the effect on nuclear condensation, and to further investigate the molecular mechanisms of formation of nuclear condensation as linearly patterned programmed cell necrosis （LPPCN）. Method Hypoxia mimetic CoC12 was used to mimic the microenvironment. B16 cells were used as the model to observe the formation of nuclear chromosomal condensation and the change of tumor cells. Wound healing assay and SRB assay were used to detect the effect of hypoxia on migration ability and proliferation of tumor cells. Western blot and RT-PCR were used to analyze the effect of hypoxia on protein and RNA expression of BCL-2. Immunofluorescence was used to determine the effect of hypoxia on the subcellular location of BCL-2. Result After 36 to 48 hours of hypoxia, nuclear staining with Hoechst 33342 revealed that there are nuclear chromosomal condensation in more than 30% of tumor cells. Most of the nuclei were abnormally shaped and deeply stained, as single nuclear condensation aggregate or several connected nuclear condensation aggregates. However, the typical apoptosis were rarely observed. Hypoxia significantly inhibited the proliferation and migration of tumor ceils compared with cells under normoxic condition. When cells were cultured in hypoxia, the expression of BCL-2 showed time-dependent expression patterns, consistent with the time of apperance of nuclear condensation. Conclusion Hypoxia can induce formation of nuclear condensation, showing the same characteristics of LPPCN. Tumor cells that survive hypoxia showed stronger migration potential. The maximum expression peak of BCL-2 induced by hypoxia may be related to formation of nuclear condensation.