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蛋白酪氨酸激酶2β介导自然杀伤细胞活性影响肺癌细胞生物学功能的体外研究

Study on the effect of PTK2B mediated NK cell killing on the malignant biological behavior of non-small cell lung cancer cells

投稿时间：2024-12-19 修订日期：2025-02-10

DOI：

中文关键词: 肺癌蛋白酪氨酸激酶2β ( PTK2B) 自然杀伤细胞杀伤作用增殖侵袭

英文关键词:Lung cancer Protein tyrosine kinase 2β (PTK2B) NK cells Killing effect proliferation Invasion

基金项目:国家自然科学基金资助项目（项目批准号：82103772）；南通市卫生健康委员会科研课题（项目编号：MSZ2023017）；南通市第一人民医院省部级以上高层次科技项目（项目编号：YPYJJZD010）

作者	单位	邮编
马春辉	南通市第一人民医院	226000
张福全	南通市第一人民医院
曹津铭	苏州大学附属第一医院
许一鸣^*	南通市第一人民医院	226000

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中文摘要:

目的：探究PTK2B介导自然杀伤细胞的杀伤作用影响肺癌细胞恶性生物学行为的作用机制研究。方法：收集2022年1月至2022年12月于南通市第一人民医院肿瘤科进行治疗的35例肺癌患者和35例健康志愿者的外周血，采用密度梯度离心对外周血单个核细胞（PBMCs）进行分离，使用磁性活化细胞分选人细胞分离试剂盒从PBMCs中分离选择细胞。应用RT-PCR技术及Western blot检测细胞中PTK2B表达水平。流式细胞术检测外周血中NKG2D、NKp30和NKp46水平，ELISA法检测外周血中IFN-γ和TNF-α水平。体外培养NK-92细胞并敲低PTK2B表达，RT-qPCR和Western blot检测NK-92细胞中PTK2B的表达。IL-2刺激NK-92细胞，ELISA检测TNF-a和IFN-γ表达。随后，IL-2刺激的NK-92细胞与A549细胞以不同的比例（10:1，5:1，2.5:1）共培养，流式细胞术和乳酸脱氢酶法评估NK-92细胞对A549细胞的毒性，CCK-8检测细胞活力，细胞克隆形成实验检测细胞增殖，Transwell检测细胞侵袭。结果：肺癌患者NK-92细胞中PTK2B的表达水平升高，且细胞上NKG2D和NKp30受体的表达水平及IFN-γ和TNF-α水平降低，但细胞活化受体NKp46的表达无显著差异。表明肺癌患者外周血中细胞杀伤活性降低。敲低PTK2B上调NK细胞活化受体NKG2D、NKp30和NKp46表达水平及细胞产生IFN-γ、颗粒酶B和穿孔素水平。此外，敲低PTK2B表达增强细胞对A549细胞的细胞毒性，使A549细胞活力、增殖和侵袭降低。表明敲低PTK2B能抑制A549细胞恶性生物学行为。结论：PTK2B通过抑制细胞的杀伤作用从而促进肺癌细胞的增殖和侵袭。

英文摘要:

Objective: To explore the mechanism of PTK2B mediated NK cell killing affecting the malignant biological behavior of non-small cell lung cancer cells.Methods: Peripheral blood of 35 patients with lung cancer and 35 healthy volunteers who were treated in the oncology department of Nantong First People's Hospital from January 2022 to December 2022 were collected. Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifuge, and NK cells were isolated and selected from PBMCs by magnetic activated cell sorting human NK cell isolation kit.The expression of PTK2B in NK cells was detected by RT-PCR and Western blot. The levels of NKG2D, NKp30 and NKp46 in peripheral blood were detected by flow cytometry, and the levels of IFN-γ and TNF-α were detected by ELISA. NK-92 cells were cultured in vitro and PTK2B expression was knocked down. The expression of PTK2B in NK-92 cells was detected by RT-qPCR and Western blot.IL-2 stimulated NK-92 cells, and the expression of TNF-a and IFN-γ was detected by ELISA. Subsequently, IL-2-stimulated NK-92 cells and A549 cells were co-cultured in different ratios (10:1, 5:1, 2.5:1). The toxicity of NK-92 cells to A549 cells was evaluated by flow cytometry and lactate dehydrogenase method, cell viability was detected by CCK-8, and cell proliferation was detected by cell clonation assay. Transwell detects cell invasion.Results: The expression level of PTK2B in NK-92 cells of lung cancer patients was increased, and the expression levels of NKG2D and NKp30 receptors, IFN-γ and TNF-α on NK cells were decreased, but there was no significant difference in the expression of NKp46 cell activating receptor. These results indicated that the killing activity of NK cells in peripheral blood of patients with lung cancer decreased. Knocking down PTK2B up-regulates the expression levels of NK cell activating receptors NKG2D, NKp30 and NKp46, and the levels of NK cell production of IFN-γ, granase B and perforin.In addition, knockdown of PTK2B expression enhanced the cytotoxicity of NK cells to A549 cells, and reduced the viability, proliferation and invasion of A549 cells. The results indicated that PTK2B knockdown could inhibit the malignant biological behavior of A549 cells.Conclusion: PTK2B can promote the proliferation, invasion and migration of Lung cancer cells by inhibiting the killing effect of NK cells.

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