| 马春辉,张福全,曹津铭,等.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[J].肿瘤学杂志,2025,31(5):401-408. |
| 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 |
| Effects of Natural Killer Cell Activity Mediated by Protein Tyrosine Kinase 2 Beta on Biological Function in Lung Cancer Cells |
| 投稿时间：2024-12-19 |
| DOI：10.11735/j.issn.1671-170X.2025.05.B005 |
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| 中文关键词: 肺肿瘤 蛋白酪氨酸激酶2β 自然杀伤细胞 杀伤作用 增殖 侵袭 |
| 英文关键词:lung neoplasms protein tyrosine kinase 2β natural killer cell killing effect proliferation invasion |
| 基金项目:国家自然科学基金资助项目（82103772）；南通市卫生健康委员会科研课题（MSZ2023017）；南通市第一人民医院省部级以上高层次科技项目（YPYJJZD010） |
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| 中文摘要: |
| 摘 要：［目的］ 探究蛋白酪氨酸激酶2β （protein tyrosine kinase 2 beta，PTK2B）介导自然杀伤细胞的杀伤作用及影响肺癌细胞恶性生物学行为的作用机制。［方法］ 收集2022年1月至12月于南通市第一人民医院就诊的35例肺癌患者和35人健康志愿者的外周血，采用密度梯度离心对外周血单核细胞进行分离，使用磁性活化细胞分选人细胞分离试剂盒从外周血单核细胞中分离选择细胞。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法检测外周血中干扰素-γ （interferon-γ，IFN-γ）和肿瘤坏死因子α（tumor necrosis factor α，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。白细胞介素2（interleukin-2，IL-2）刺激NK-92细胞，ELISA检测TNF-α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］ 与对照组相比，肺癌患者中PTK2B mRNA 和蛋白水平升高（1.15±0.51 vs 3.51±1.25，0.40±0.10 vs 0.82±0.15）（P均＜0.001），NKG2D（48.86±8.03 vs 27.26±11.56，P＜0.001）、NKp30（23.77±7.9 vs 16.10±7.03，P＜0.001）、IFN-γ［（14.25±2.29）ng/mL vs （4.17±0.87）ng/mL，P＜0.001］和TNF-α［（212.50±22.57）pg/mL vs （59.19±17.94）pg/mL，P＜0.001］水平降低，但细胞活化受体NKp46表达无显著性差异（16.96±4.88 vs 18.20±6.73，P＞0.05）。敲低PTK2B上调 NKG2D和NKp30、NKp46表达水平，以及增加NK细胞产生IFN-γ、颗粒酶B和穿孔素水平。 敲低PTK2B表达增强细胞对A549细胞的细胞毒性、 细胞活力、增殖和侵袭降低，并能抑制A549细胞恶性生物学行为。［结论］ PTK2B通过抑制自然杀伤细胞的杀伤作用，从而促进肺癌细胞的增殖和侵袭。 |
| 英文摘要: |
| Abstract： ［Objective］ To explore the mechanism of protein tyrosine kinase 2 beta (PTK2B) mediating the killing effect of natural killer(NK) cell and the malignant biological behavior of lung cancer cells. ［Methods］ Peripheral blood samples were collected from 35 lung cancer patients and 35 healthy volunteers who were treated in Nantong First People’s Hospital from January to December 2022. Peripheral blood monocytes were isolated by density gradient centrifugation， and cells were isolated and selected from peripheral blood monocytes by magnetic activated cell sorting human cell separation 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 interferon-γ(IFN-γ) and tumor necrosis factor α(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-α 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 PTK2B mRNA and PTK2B protein expression in lung cancer patients was increased(1.15±0.51 vs 3.51±1.25， 0.40±0.10 vs 0.82±0.15， all P<0.001)； and NKG2D(48.86±8.03 vs 27.26±11.56， P<0.001)， NKp30 (23.77±7.9 vs 16.10±7.03， P<0.001)， IFN-γ［(14.25±2.29) ng/mL vs (4.17±0.87) ng/mL， P<0.001］ and TNF-α［(212.50±22.57) pg/mL vs (59.19±17.94) pg/mL， P<0.001］ decreased， but there was no significant difference in the expression of NKp46 (16.96±4.88 vs 18.20±6.73， P>0.05). Knockdown of PTK2B up-regulated NK cell activation receptors NKG2D， NKp30 and NKp46， and IFN-γ， granzyme B and perforin in NK cell. In addition， knockdown of PTK2B expression enhanced the cytotoxicity， viability， proliferation and invasion. These results indicated that knockdown of PTK2B 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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