人淋巴细胞来源的外泌体mRNAs作为辐射生物标志物的筛选与验证

doi:10.3969/j.issn.1004-616x.2026.01.007

摘要/Abstract

摘要： 目的：探讨电离辐射对人外周血B淋巴细胞系(AHH-1细胞)来源外泌体中mRNAs表达谱的影响，及其差异表达基因作为辐射生物标志物的可能性。方法：本研究以AHH-1细胞为模型，分别收集对照组和2、5 Gy ⁶⁰Co γ射线照射后24 h的细胞培养上清，通过超高速差速离心法分离外泌体，并利用纳米颗粒追踪技术、透射电镜和Western blot方法对外泌体粒径、形态及表面标志蛋白(CD63与TSG101)进行检测。利用表达谱芯片筛选差异表达mRNAs，并进行GO与KEGG富集分析。为进一步探究候选基因的剂量效应关系和时间响应范围，采用0、1、2、4、6 Gy γ射线照射AHH-1细胞，并分别于照射后24和48 h收集外泌体样本，提取外泌体RNA，通过实时荧光定量PCR技术检测候选基因的mRNA相对表达水平。结果：与对照组相比，AHH-1细胞经2 Gy γ射线照射24 h后，外泌体中共鉴定出197个差异表达的mRNAs，其中184个表达上调、13个表达下调；经5 Gy γ射线照射后共鉴定出192个差异表达的mRNAs，其中170个表达上调、22个表达下调。生物信息学分析显示，这些表达变化的mRNAs可能通过参与TGF-β信号通路、ErbB信号通路等影响细胞增殖、凋亡。实时荧光定量PCR分析结果显示，0～6 Gy γ射线照射后24 h，ZNFX1、DDB2、SPDYE2B和CHD3 mRNA均呈现出显著上调的趋势；LENG8、FBXW7、AKAP1及ZGRF1 mRNA在0～4Gy γ射线照射后48 h显著上调，其中4 Gy γ射线照射后48 h的LENG8与FBXW7 mRNA表达水平约为对照组的3倍。结论：电离辐射可诱导AHH-1细胞来源外泌体的mRNAs表达谱发生显著改变，筛选出的差异表达基因(ZNFX1、DDB2、SPDYE2B、CHD3、LENG8、FBXW7、AKAP1和ZGRF1)具有成为电离辐射生物标志物的潜力。

关键词: AHH-1细胞系, 人外周血B淋巴细胞, 外泌体, mRNAs, 电离辐射, 基因表达, 生物标志物

Abstract: OBJECTIVE： To investigate effects of ionizing radiation on the mRNA expression profile in exosomes from the AHH-1 human peripheral blood B lymphoblast cell line and to investigate usefulness of the profile as radiation biomarkers. METHODS： Using the AHH-1 cell line as a model， culture supernatants were collected from control cells and cells exposed to 2 Gy or 5 Gy of γ-rays at 24 h postirradiation. Exosomes were isolated by differential ultracentrifugation and characterized via nanoparticle tracking analysis for size distribution，transmission electron microscopy for morphology，and Western blotting for the surface markers CD63 and TSG101. Differentially expressed mRNAs were screened using expression microarray and subsequently subjected to GO and KEGG enrichment analyses. To further examine the dose- and time-dependent responses of candidate genes， AHH-1 cells were irradiated with 0， 1， 2， 4， or 6 Gy of γ-rays，and exosomes were collected at 24 h and 48 h postirradiation. Exosomal RNA was extracted，and the relative expression levels of selected mRNAs were validated by quantitative realtime PCR. RESULTS： Compared to the control group，a total of 197 differentially expressed mRNAs were identified in the exosomes from the 2 Gy irradiated cells，with 184 being upregulated and 13 downregulated. Following 5 Gy irradiation， 192 differentially expressed mRNAs were identified， comprising 170 upregulated and 22 downregulated transcripts. Bioinformatic analysis indicated that these altered mRNAs influenced cellular processes such as proliferation and apoptosis by participating in pathways including the TGF-β and ErbB signaling pathways. Real-time quantitative PCR analysis of the relative expression changes of candidate genes revealed that at 24 h after 0-6 Gy γ-rays irradiation， ZNFX1， DDB2， SPDYE2B， and CHD3 mRNA exhibited a significant upregulation trend. At 48 h after 4 Gy γ-rays irradiation， LENG8， FBXW7， AKAP1， and ZGRF1 mRNA were significantly upregulated. Notably，the expression levels of LENG8 and FBXW7 mRNA at 48 h after 4 Gy irradiation were approximately three times that of the control group. CONCLUSION： Ionizing radiation significantly altered mRNA expression profile in exosomes derived from AHH-1 cells. The differentially expressed genes identified (ZNFX1， DDB2， SPDYE2B， CHD3， LENG8， FBXW7， AKAP1， and ZGRF1) showed potential as biomarkers for ionizing radiation exposure.

Key words: AHH-1 cell line, human peripheral blood B lymphocyte, exosomes, mRNAs, radiation exposure, gene expression, biomarkers

中图分类号:

Q691.5

周瑞霞, 刘俊, 陆雪, 蔡恬静, 闫娟, 刘青杰, 李爽. 人淋巴细胞来源的外泌体mRNAs作为辐射生物标志物的筛选与验证[J]. 癌变·畸变·突变, 2026, 38(1): 41-47,70.

ZHOU Ruixia, LIU Jun, LU Xue, CAI Tianjing, YAN Juan, LIU Qingjie, LI Shuang. Preliminary validation of exosome mRNAs from human lymphocytes as radiation biomarkers[J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2026, 38(1): 41-47,70.

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