X射线对A375细胞凋亡和增殖影响研究

Investigation of the Impact of X-Ray on Apoptosis and Proliferation in A375 Cells

摘要: 目的：本研究旨在探讨X射线对人黑色素瘤细胞A375的放射损伤效应及其分子机制。方法：采用0 Gy、2 Gy、4 Gy、6 Gy、8 Gy、10 Gy的X射线梯度辐射A375细胞，辐射后48小时，利用CCK-8法检测细胞活性，CFSE标记法检测细胞增殖，Annexin V-FITC/PI双标法检测细胞凋亡，并确定半数致死剂量(LD50)。采用LD50剂量X射线处理A375细胞，辐射后培养48小时后，提取细胞总RNA并进行转录组测序。通过差异基因分析及GO和KEGG富集分析解析分子机制，并对关键差异基因进行qRT-PCR验证。结果：与0 Gy组相比，2 Gy、4 Gy、6 Gy、8 Gy和10 Gy辐射后48小时，细胞凋亡率分别为27.02%、41.42%、58.25%、71.73%和82.55%，增殖率分别为31.57%、25.67%、21.37%、11.13%和7.68%。转录组分析显示，X射线辐射处理组有254个显著差异基因，其中上调234个，下调20个。GO和KEGG分析表明，上调差异基因显著富集于炎症反应、免疫应答及NF-κB信号通路等凋亡相关功能与通路；下调差异基因显著富集于细胞周期调控及DNA损伤修复相关通路。qRT-PCR验证结果与转录组数据一致，进一步证实了实验结果的可靠性。结论：X射线通过激活凋亡信号通路、抑制细胞周期及DNA修复通路调控A375细胞，该结果为放射治疗提供了一定的理论依据。

Abstract: Objective: This study aims to investigate the radiological damage effects of X-ray irradiation on human melanoma A375 cells and explore the underlying molecular mechanisms. Methods: A375 cells were irradiated with X-rays at doses of 0 Gy, 2 Gy, 4 Gy, 6 Gy, 8 Gy, and 10 Gy. After 48 hours, cell viability was assessed using the CCK-8 assay, cell proliferation was measured by CFSE labeling, and apoptosis was detected using Annexin V-FITC/PI double staining. The median lethal dose (LD50) was determined. Subsequently, A375 cells were treated with X-rays at the LD50 dose, and total RNA was extracted after 48 hours for transcriptome sequencing. Differential gene expression analysis, along with GO and KEGG enrichment analyses, was performed to elucidate the molecular mechanisms. Key differentially expressed genes were further validated by qRT-PCR. Results: Compared to the 0 Gy group, the apoptosis rates at 48 hours post-irradiation with 2 Gy, 4 Gy, 6 Gy, 8 Gy, and 10 Gy were 27.02%, 41.42%, 58.25%, 71.73%, and 82.55%, respectively, while the proliferation rates were 31.57%, 25.67%, 21.37%, 11.13%, and 7.68%, respectively. Transcriptome analysis revealed 254 significantly Differentially expressed genes (DEGs) in the X-ray irradiated groups, including 234 upregulated and 20 downregulated genes. GO and KEGG analyses indicated that the upregulated DEGs were significantly enriched in apoptosis-related functions and pathways, such as inflammatory response, immune response, and the NF-κB signaling pathway, while the downregulated DEGs were significantly enriched in pathways related to cell cycle regulation and DNA damage repair. The qRT-PCR validation results were consistent with the transcriptome data, further confirming the reliability of the identified DEGs. Conclusion: X-ray irradiation exerts its regulatory effects on A375 cells by activating apoptotic signaling pathways while concurrently inhibiting cell cycle progression and DNA repair mechanisms, thereby providing a theoretical foundation for the application of X-ray-based radiotherapy in melanoma treatment.