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目的 :通过构建肿瘤细胞失巢培养模型,探讨p75NTR ICD对肿瘤细胞失巢凋亡抵抗能力的影响及其相关信号通路的激活情况。方法:采用免疫组织化学方法观察p75NTR ICD在口腔鳞状细胞癌组织中的表达,Western免疫印迹方法检测高转移和低转移潜能口腔鳞癌细胞系中p75NTR ICD的表达。通过poly-HEMA(聚甲基丙烯酸羟乙基酯)包被培养皿,建立肿瘤细胞失巢培养模型。通过质粒转染方法,在细胞中过表达p75NTR ICD。采用流式细胞术检测肿瘤细胞在失巢培养条件下的凋亡水平,通过激光共聚焦显微镜及Western免疫印迹实验检测肿瘤细胞失巢培养模型中相关信号通路的激活情况。采用SPSS.22软件包对数据进行统计学分析。结果:发生淋巴结转移的口腔鳞状细胞癌原发灶组织标本中,p75NTR ICD定位于胞质中;未发生淋巴结转移的口腔鳞状细胞癌原发灶组织标本中,p75NTR ICD定位于胞膜上。高转移潜能HN-12细胞系中p75NTR ICD高表达,低转移潜能HN-4细胞中p75NTR ICD低表达,外源性过表达p75NTR ICD可降低HN-4细胞的失巢凋亡水平。与HN-4细胞相比,HN-12失巢培养后形成更大而圆的小球。HN-12细胞失巢凋亡水平低于HN-4细胞,高转移潜能HN-12细胞失巢培养后激活NF-κB,低转移潜能HN-4细胞失巢培养,NF-κB未能被激活。结论:p75NTR ICD在肿瘤细胞中的定位与肿瘤是否发生淋巴结转移相关。p75NTR ICD高表达肿瘤细胞具有失巢凋亡抵抗能力,p75NTR ICD通过激活NF-κB信号通路促进肿瘤细胞失巢凋亡抵抗。
OBJECTIVE: To investigate the effect of p75NTR ICD on the resistance of tumor cells to anoikis and the activation of related signaling pathways by constructing an in vitro model of tumor cells. Methods: Immunohistochemistry was used to observe the expression of p75NTR ICD in oral squamous cell carcinoma tissues. Western blotting was used to detect the expression of p75NTR ICD in high metastasis and low metastatic potential squamous cell carcinoma. Petri dishes were coated with poly-HEMA (polyhydroxyethylmethacrylate) to establish a model of tumor cell de-nesting. The p75NTR ICD was overexpressed in the cells by the plasmid transfection method. Flow cytometry was used to detect the apoptosis of tumor cells under anaerobic culture conditions. The activation of related signaling pathways was detected by laser scanning confocal microscopy and Western blotting. SPSS.22 software package for statistical analysis of the data. Results: The p75NTR ICD was localized in the cytoplasm in primary squamous cell carcinoma tissues with lymph node metastasis. In primary squamous cell carcinoma without lymph node metastasis, the p75NTR ICD was located on the membrane . The overexpression p75NTR ICD of p75NTR ICD in HN-12 cell line with high metastatic potentiality and low metastatic potential HN-4 cell line with overexpression of p75NTR ICD can reduce the level of anoikis in HN-4 cells. Compared to HN-4 cells, HN-12 forms a larger, round ball after de-nesting. The level of anoikis in HN-12 cells was lower than that in HN-4 cells. The high metastatic potential HN-12 cells activated NF-κB and the low metastatic potential HN-4 cells in vitro and inactivated by NF-κB . Conclusion: The localization of p75NTR ICD in tumor cells is related to the occurrence of lymph node metastasis. p75NTR ICD high expression of tumor cells with anoikis resistance, p75NTR ICD through the activation of NF-κB signaling pathway to promote tumor cell anoikis resistance.