多西他赛诱导的非小细胞肺癌NCI-H1299多倍体肿瘤巨细胞衰老、干性和再增殖的特征研究

doi:10.3760/cma.j.cn371439-20240727-00114

摘要/Abstract

摘要：

目的探讨多西他赛（Doc）诱导的非小细胞肺癌多倍体肿瘤巨细胞细胞衰老、肿瘤干细胞和细胞增殖特征，分析多倍体肿瘤巨细胞在肿瘤复发中的潜在作用。方法二甲基亚砜（DMSO）或100 nmol/L Doc处理非小细胞肺癌NCI-H1299细胞24 h，更换新鲜的完全培养基继续培养3 d，分别记为对照组和Doc组。显微镜观察细胞生长状态，免疫荧光染色检测细胞核形态，流式细胞术检测DNA含量，台盼蓝染色统计细胞密度，衰老相关的β-半乳糖苷酶染色检测β-半乳糖苷酶活性，实时荧光定量PCR检测转录因子Krüppel样因子4 （KLF4）和NANOG的mRNA表达水平，Doc组细胞长期培养观察多倍体肿瘤巨细胞生长情况。结果 Doc处理NCI-H1299后，细胞体积变大，细胞核体积变大。Doc组多倍体百分比为45.80%±1.73%，明显高于对照组的4.83%±0.72%，差异具有统计学意义（t=-29.01，P<0.001）。Doc组细胞密度为（8.18±0.54）×10⁴/cm²，明显高于对照组的（0.75±0.08）×10⁴/cm²，差异具有统计学意义（t=23.55，P<0.001）。Doc组细胞衰老相关的β-半乳糖苷酶活性增加，统计阳性染色（蓝色）细胞百分比为56.21%±3.38%，明显高于对照组的4.20%±0.79%，差异具有统计学意义（t=-47.37，P<0.001）。与对照组（1.00±0.16）相比，Doc组KLF4的mRNA表达水平（2.47±0.18）较高，差异具有统计学意义（t=-10.74，P<0.001）。与对照组（0.99±0.07）相比，Doc 组NANOG的mRNA表达水平（5.58±0.26）较高，差异具有统计学意义（t=-30.09，P<0.001）。Doc组细胞继续培养5 d，细胞保持大细胞状态，视野内细胞较少；继续培养7 d，大细胞周围出现小的子代细胞，视野内细胞仍较少；继续培养13 d，子代细胞数量明显增多；继续培养15 d，子代细胞最终占满整个视野。结论 Doc诱导非小细胞肺癌NCI-H1299细胞产生多倍体肿瘤巨细胞，表现出衰老、干性和再增殖特征，可能促进肿瘤复发。

关键词: 肺肿瘤, 多倍体, 细胞衰老, 肿瘤干细胞, 细胞增殖, 肿瘤复发

Abstract:

Objective To investigate the characteristics of cellular senescence， neoplastic stem cell and cell proliferation of polyploid giant cancer cells induced by Docetaxel （Doc） in non-small cell lung cancer， so as to analyze the potential role of polyploid giant cancer cells in neoplasm recurrence. Methods Non-small cell lung cancer NCI-H1299 cells were treated with dimethyl sulfoxide （DMSO） or 100 nmol/L Doc for 24 h， and then cultured for another 3 d after replacing with fresh complete medium. The cells were recorded as control group and Doc group， respectively. The cell growth was observed under a microscopy. The nuclear morphology was detected by immunofluorescence staining. The DNA content was detected by flow cytometry. The cell density was counted by trypan blue staining. A senescence-associated β-galactosidase staining kit was used to detect the β-galactosidase activity. The mRNA expression levels of the transcription factors Krüppel-like factor 4 （KLF4） and NANOG were detected by real-time quantitative polymerase chain reaction （PCR）. The cells in the Doc group were cultured for a long time to observe the growth of polyploid giant cancer cells. Results After Doc treatment， the cell and nuclear volume were increased in NCI-H1299 cells. The percentage of polyploidy in the Doc group was 45.80%±1.73%， which was significantly higher than that in the control group （4.83%±0.72%）， with a statistically significant difference （t=-29.01， P<0.001）. The cell density in the Doc group was （8.18±0.54）×10⁴/cm²， which was significantly higher than that in the control group [（0.75±0.08）×10⁴/cm²]， with a statistically significant difference （t=23.55， P<0.001）. The senescence-associated β-galactosidase activity was significantly increased and the percentage of positive cells （blue coloring） was 56.21%±3.38% in the Doc group， which was significantly higher than that in the control group （4.20%±0.79%）， with a statistically significant difference （t=-47.37， P<0.001）. Compared with the control group （1.00±0.16）， the mRNA expression level of KLF4 was significantly higher in the Doc group （2.47±0.18）， with a statistically significant difference （t=-10.74， P<0.001）. Compared with the control group （0.99±0.07）， the mRNA expression level of NANOG was significantly higher in the Doc group （5.58±0.26）， with a statistically significant difference （t=-30.09， P<0.001）. After being cultured for another 5 d， the cells in the Doc group maintained a large cellular state and there were less cells in the visual field. After being cultured for 7 d， a few small daughter cells appeared around the large cells and there were still few cells in the visual field. After being cultured for 13 d， the number of daughter cells was significantly increased. And the daughter cells finally occupied the entire field of view by 15 d. Conclusion Doc induces non-small cell lung cancer NCI-H1299 cells to generate polyploid giant cancer cells. These cells exhibit characteristics of the senescence， stemness and reproliferation， which may contribute to neoplasm recurrence.

Key words: Lung neoplasms, Polyploid, Cellular senescence, Neoplastic stem cell, Cell proliferation, Neoplasm recurrence

赵松, 王丽丽, 欧阳明玥, 邢思宁, 杨紫恩, 于卉影. 多西他赛诱导的非小细胞肺癌NCI-H1299多倍体肿瘤巨细胞衰老、干性和再增殖的特征研究[J]. 国际肿瘤学杂志, 2024, 51(11): 673-677.

Zhao Song, Wang Lili, Ouyang Mingyue, Xing Sining, Yang Zi'en, Yu Huiying. Study on the characteristics of senescence， stemness and reproliferation of non-small cell lung cancer NCI-H1299 polyploid giant cancer cells induced by Docetaxel[J]. Journal of International Oncology, 2024, 51(11): 673-677.

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基因名称	正向引物（5'-3'）	反向引物（5'-3'）
KLF4	TGGACCCCCTCTCAGCAAT	CAGCACTTCCTCAAGACCCAG
NANOG	TTCCACCAGTCCCAAAGG	AGGTTCAGGATGTTGGAGAGTT
β-actin	TGAAGGTGACAGCAGTCGGTT	AAGTGGGGTGGCTTTTAGGAT