{"title":"使用外周全血进行基于基因表达的生物剂量测定的简化方案。","authors":"Shuchi Bhagi, Sudhir Chandna","doi":"10.1080/09553002.2023.2231531","DOIUrl":null,"url":null,"abstract":"<p><strong>Purpose: </strong>Assessing alterations in the expression of radiation-responsive genes in peripheral blood cells is considered a promising approach for high-throughput radiation biodosimetry. However, optimization of conditions for storage and transport of blood samples would be critical for obtaining reliable results. Recent studies involved the incubation of isolated peripheral blood mononuclear cells (in cell culture medium) and/or use of RNA stabilizing agents for sample storage, immediately after the <i>ex vivo</i> irradiation of whole blood. We used a simpler protocol by incubating undiluted peripheral whole blood without any RNA stabilizing agent, and studied the impact of storage temperature and incubation time on the expression levels of 19 known radiation responsive genes.</p><p><strong>Materials & methods: </strong>Peripheral whole blood was γ-irradiated <i>ex vivo</i> at room temperature at low (0.5 Gy), moderate (1 Gy, 2 Gy) and high (4 Gy) doses and immediately incubated at two different temperatures at 4 °C or 37 °C for 2h, 4h and 24 h. Using qRT-PCR, mRNA expression levels of <i>CDKN1A, DDB2, GADD45A, FDXR, BAX, BBC3, MYC, PCNA, XPC, ZMAT3, AEN, TRIAP1, CCNG1, RPS27L, CD70, EI24, C12orf5, TNFRSF10B, ASCC3</i> were analyzed at respective time-points and compared with the sham-irradiated controls.</p><p><strong>Results: </strong>Transcriptional responses of all 19 genes did not alter significantly upon incubation of whole blood samples at 4 °C, as compared to untreated controls. However, incubation at 37 °C for 24 h resulted in significant radiation-induced overexpression in 14 out of the 19 genes analyzed (except <i>CDKN1A, BBC3, MYC, CD 70</i> and <i>EI24</i>). Detailed patterns during incubation at 37 °C revealed time-dependent up-regulation of these genes, with <i>DDB2</i> and <i>FDXR</i> showing significant up-regulation both at 4 and 24 h with the highest fold-change observed.</p><p><strong>Conclusion: </strong>Overall, the undiluted whole blood incubated at 37 °C for 24 h was found to elicit most optimal transcriptional response in the genes studied, with most profound overexpression of <i>DDB2</i> and <i>FDXR</i>. We propose that sample storage/transport/post-transit incubation at the physiological temperature for up to 24 h may enhance the sensitivity of gene expression based biodosimetry and facilitate its usage for triage application.</p>","PeriodicalId":14261,"journal":{"name":"International Journal of Radiation Biology","volume":null,"pages":null},"PeriodicalIF":2.1000,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"A simplified protocol for gene expression-based biological dosimetry using peripheral whole blood.\",\"authors\":\"Shuchi Bhagi, Sudhir Chandna\",\"doi\":\"10.1080/09553002.2023.2231531\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><strong>Purpose: </strong>Assessing alterations in the expression of radiation-responsive genes in peripheral blood cells is considered a promising approach for high-throughput radiation biodosimetry. However, optimization of conditions for storage and transport of blood samples would be critical for obtaining reliable results. Recent studies involved the incubation of isolated peripheral blood mononuclear cells (in cell culture medium) and/or use of RNA stabilizing agents for sample storage, immediately after the <i>ex vivo</i> irradiation of whole blood. We used a simpler protocol by incubating undiluted peripheral whole blood without any RNA stabilizing agent, and studied the impact of storage temperature and incubation time on the expression levels of 19 known radiation responsive genes.</p><p><strong>Materials & methods: </strong>Peripheral whole blood was γ-irradiated <i>ex vivo</i> at room temperature at low (0.5 Gy), moderate (1 Gy, 2 Gy) and high (4 Gy) doses and immediately incubated at two different temperatures at 4 °C or 37 °C for 2h, 4h and 24 h. Using qRT-PCR, mRNA expression levels of <i>CDKN1A, DDB2, GADD45A, FDXR, BAX, BBC3, MYC, PCNA, XPC, ZMAT3, AEN, TRIAP1, CCNG1, RPS27L, CD70, EI24, C12orf5, TNFRSF10B, ASCC3</i> were analyzed at respective time-points and compared with the sham-irradiated controls.</p><p><strong>Results: </strong>Transcriptional responses of all 19 genes did not alter significantly upon incubation of whole blood samples at 4 °C, as compared to untreated controls. However, incubation at 37 °C for 24 h resulted in significant radiation-induced overexpression in 14 out of the 19 genes analyzed (except <i>CDKN1A, BBC3, MYC, CD 70</i> and <i>EI24</i>). Detailed patterns during incubation at 37 °C revealed time-dependent up-regulation of these genes, with <i>DDB2</i> and <i>FDXR</i> showing significant up-regulation both at 4 and 24 h with the highest fold-change observed.</p><p><strong>Conclusion: </strong>Overall, the undiluted whole blood incubated at 37 °C for 24 h was found to elicit most optimal transcriptional response in the genes studied, with most profound overexpression of <i>DDB2</i> and <i>FDXR</i>. We propose that sample storage/transport/post-transit incubation at the physiological temperature for up to 24 h may enhance the sensitivity of gene expression based biodosimetry and facilitate its usage for triage application.</p>\",\"PeriodicalId\":14261,\"journal\":{\"name\":\"International Journal of Radiation Biology\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":2.1000,\"publicationDate\":\"2023-01-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"International Journal of Radiation Biology\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://doi.org/10.1080/09553002.2023.2231531\",\"RegionNum\":4,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"2023/7/12 0:00:00\",\"PubModel\":\"Epub\",\"JCR\":\"Q2\",\"JCRName\":\"BIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"International Journal of Radiation Biology","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.1080/09553002.2023.2231531","RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2023/7/12 0:00:00","PubModel":"Epub","JCR":"Q2","JCRName":"BIOLOGY","Score":null,"Total":0}
引用次数: 0
摘要
目的:评估外周血细胞中辐射反应基因表达的变化被认为是高通量辐射生物剂量测定的一种有前途的方法。然而,优化血液样本的储存和运输条件对于获得可靠的结果至关重要。最近的研究涉及在全血离体照射后立即培养分离的外周血单核细胞(在细胞培养基中)和/或使用RNA稳定剂进行样品储存。我们使用了一种更简单的方案,在没有任何RNA稳定剂的情况下孵育未稀释的外周全血,并研究了储存温度和孵育时间对19个已知辐射反应基因表达水平的影响。材料与方法:外周血经γ射线低(0.5 Gy),中度(1 Gy,2 Gy)和高(4 Gy)剂量,并立即在两个不同的温度下在4 °C或37 °C持续2小时、4小时和24小时 h.使用qRT-PCR,在各个时间点分析CDKN1A、DDB2、GADD45A、FDXR、BAX、BBC3、MYC、PCNA、XPC、ZMAT3、AEN、TRIAP1、CCNG1、RPS27L、CD70、EI24、C12orf5、TNFRSF10B、ASCC3的mRNA表达水平,并与假照射对照进行比较。结果:所有19个基因的转录反应在4 °C,与未经处理的对照组相比。然而,37岁时孵化 °C 24 h导致所分析的19个基因中的14个基因(CDKN1A、BBC3、MYC、CD 70和EI24除外)出现显著的辐射诱导过表达。37培养期间的详细模式 °C显示这些基因的时间依赖性上调,DDB2和FDXR在4和24时均显示显著上调 h,观察到最高的倍数变化。结论:总的来说,未稀释的全血在37 °C 24 发现h在所研究的基因中引发最优化的转录反应,DDB2和FDXR的过度表达最为严重。我们建议样品在生理温度下储存/运输/运输后培养长达24小时 h可以增强基于基因表达的生物剂量测定的敏感性,并促进其在分诊应用中的应用。
A simplified protocol for gene expression-based biological dosimetry using peripheral whole blood.
Purpose: Assessing alterations in the expression of radiation-responsive genes in peripheral blood cells is considered a promising approach for high-throughput radiation biodosimetry. However, optimization of conditions for storage and transport of blood samples would be critical for obtaining reliable results. Recent studies involved the incubation of isolated peripheral blood mononuclear cells (in cell culture medium) and/or use of RNA stabilizing agents for sample storage, immediately after the ex vivo irradiation of whole blood. We used a simpler protocol by incubating undiluted peripheral whole blood without any RNA stabilizing agent, and studied the impact of storage temperature and incubation time on the expression levels of 19 known radiation responsive genes.
Materials & methods: Peripheral whole blood was γ-irradiated ex vivo at room temperature at low (0.5 Gy), moderate (1 Gy, 2 Gy) and high (4 Gy) doses and immediately incubated at two different temperatures at 4 °C or 37 °C for 2h, 4h and 24 h. Using qRT-PCR, mRNA expression levels of CDKN1A, DDB2, GADD45A, FDXR, BAX, BBC3, MYC, PCNA, XPC, ZMAT3, AEN, TRIAP1, CCNG1, RPS27L, CD70, EI24, C12orf5, TNFRSF10B, ASCC3 were analyzed at respective time-points and compared with the sham-irradiated controls.
Results: Transcriptional responses of all 19 genes did not alter significantly upon incubation of whole blood samples at 4 °C, as compared to untreated controls. However, incubation at 37 °C for 24 h resulted in significant radiation-induced overexpression in 14 out of the 19 genes analyzed (except CDKN1A, BBC3, MYC, CD 70 and EI24). Detailed patterns during incubation at 37 °C revealed time-dependent up-regulation of these genes, with DDB2 and FDXR showing significant up-regulation both at 4 and 24 h with the highest fold-change observed.
Conclusion: Overall, the undiluted whole blood incubated at 37 °C for 24 h was found to elicit most optimal transcriptional response in the genes studied, with most profound overexpression of DDB2 and FDXR. We propose that sample storage/transport/post-transit incubation at the physiological temperature for up to 24 h may enhance the sensitivity of gene expression based biodosimetry and facilitate its usage for triage application.
期刊介绍:
The International Journal of Radiation Biology publishes original papers, reviews, current topic articles, technical notes/reports, and meeting reports on the effects of ionizing, UV and visible radiation, accelerated particles, electromagnetic fields, ultrasound, heat and related modalities. The focus is on the biological effects of such radiations: from radiation chemistry to the spectrum of responses of living organisms and underlying mechanisms, including genetic abnormalities, repair phenomena, cell death, dose modifying agents and tissue responses. Application of basic studies to medical uses of radiation extends the coverage to practical problems such as physical and chemical adjuvants which improve the effectiveness of radiation in cancer therapy. Assessment of the hazards of low doses of radiation is also considered.