| 薛珊,黄丽婷,任小雨,等.共培养体系下 M1型巨噬细胞对宫颈癌 HeLa细胞的干预作用[J].安徽医药,2025,29(5):1029-1036. |
| 共培养体系下 M1型巨噬细胞对宫颈癌 HeLa细胞的干预作用 |
| Intervention effect of M1 macrophages on HeLa cells in co-culture system |
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| DOI：10.3969/j.issn.1009-6469.2025.05.034 |
| 中文关键词: HeLa细胞 M1型巨噬细胞 数据非依赖采集蛋白质谱 免疫干预 |
| 英文关键词: HeLa cells M1 macrophages Data-independent acquisition protein mass spectrometry Immune intervention |
| 基金项目:韶关市卫生健康科研项目( Y21293) |
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| 中文摘要: |
| 目的探讨共培养体系下 M1型巨噬细胞对宫颈癌细胞 HeLa产生的干预作用。方法 2023年 1―5月，建立 U937细胞诱导的 M1型巨噬细胞和 HeLa细胞的共培养模型，共培养 8d后，对共培养组和常规培养组的 HeLa细胞进行数据非依赖采集(DIA)蛋白质谱检测，比较后得到两组细胞的差异蛋白，并对差异蛋白进行基因本体(GO)功能注释分析、京都基因与基因组百科全书( KEGG)通路注释分析以及蛋白质相互作用( PPI)分析以获取其中的关键蛋白。对两组细胞进行细胞增殖、周期、凋亡等细胞生物学行为的检测，挑选 6个蛋白进行蛋白质印迹法验证蛋白质谱的结果。结果构建了 M1巨噬细胞和 HeLa细胞共培养平台及长时程培养的模型；通过 DIA蛋白质谱检测，获得了 1 417个差异蛋白，共培养组相比对照组，表达下调的蛋白 1 096个，上调的蛋白 321个。通过对差异蛋白的生物信息学分析，得到可能具有重要作用的 10个关键蛋白。细胞生物学检测显示，共培养 8d后:细胞计数试剂盒 8(CCK-8)法检测细胞增殖，在 24、48、72、96 h四个时间点，共培养组细胞 A450分别为 0.54± 0.02，0.71±0.01，1.10±0.02，1.53±0.02；对照组细胞 A450分别为 0.71±0.02，1.06±0.03，2.03±0.07，3.08±0.06；共培养组的 HeLa细胞相比常规培养组，增殖减缓( P<0.001)。流式细胞术检测细胞周期和凋亡，共培养组 HeLa细胞总凋亡率高于对照组［(22.87±2.11)%比( 9.66±0.43)%，P<0.001］G0/G1期细胞比例低于对照组［(44.93±6.57)%比( 63.33±2.00)%，P<0.01］G2/M期细胞比例高于对照组［( 10.04±1.44)%比6±1.51)%，P<0.01］表明共培养组 HeLa细胞凋亡增加、发生 G2/M期阻。选择 6个相关蛋白 STAT3、KPNA2、CDK1、GLUT1、PCNA和 p16 INK4a行 WB实验检测，其表达变化与蛋白质谱结果一致。结论建立(2.6，滞，进，的 M1型巨噬细胞与 HeLa细胞共培养模型对长时、动态研究免疫细胞对肿瘤细胞的影响具有重要意义。 |
| 英文摘要: |
| Objective To investigate the intervention effect of M1 macrophages on cervical cancer cells HeLa in co-culture system. Methods A co-culture model of M1 macrophages and HeLa cells was established from January to May 2023. After 8 days of co-cul-ture, the HeLa cells in the co-culture group and the conventional culture group were detected by data independent acquisition (DIA)protein mass spectrometry, and the differentially expressed proteins of the two groups were obtained after comparison. The differentiallyexpressed proteins were analyzed by Gene Ontology (GO) functional annotation analysis, Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway annotation analysis, and protein-protein interaction (PPI) analysis to obtain the key proteins. The cellular biology de-tections such as cell proliferation, cell cycle, and apoptosis were detected in the two groups of HeLa cells, and six proteins were select-ed for Western Blotting (WB) to verify the results of the protein mass spectrometry.Results A co-culture and long-term culture model of M1 macrophages and HeLa cells were established. Through DIA protein mass spectrometry, 1 417 differentially expressed proteinswere obtained. Compared to the control group, the co-culture group had 1 096 down-regulated proteins and 321 up-regulated proteins.Through bioinformatics analysis of the differentially expressed proteins, 10 key proteins with potential important roles were identified.Cellular biology detection after 8 days of co-culture: CCK-8 assay showed that the proliferation of HeLa cells in the co-culture group was significantly slower than in the control group (P<0.001), values of absorbance in 450nm of the co-culture group at 24 h, 48 h, 72 h,and 96 h were 0.54±0.02, 0.71±0.01, 1.10±0.02, and 1.53±0.02, as those of the control group were 0.71±0.02,1.06±0.03,2.03±0.07,3.08±0.06. The total apoptosis rate of HeLa cells in the co-culture group was higher than that in the control group [(22.87±2.11)% vs. (9.66±0.43)%, P<0.001], and the proportion of G0/G1 phase cells was lower than that in the control group [(44.93±6.57)% vs. (63.33±2.00)%,P<0.01], while the proportion of G2/M phase cells was higher than that in the control group [(10.04±1.44)% vs. (2.66±1.51)%, P <0.01], indicating increased apoptosis and G2/M phase arrest in HeLa cells in the co-culture group. Six related proteins, includingSTAT3, KPNA2, CDK1, GLUT1, PCNA, and p16 INK4a, were selected for WB detection, and their expression changes were consistentwith the data of the protein mass spectrometry.Conclusion The established co-culture model of M1 macrophages and HeLa cells is of great significance for long-term and dynamic studies on the effects of immune cells on tumor cells. |
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