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首页> 外文期刊>Biomaterials Science >3D encapsulation and inflammatory licensing of mesenchymal stromal cells alter the expression of common reference genes used in real-time RT-qPCR
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3D encapsulation and inflammatory licensing of mesenchymal stromal cells alter the expression of common reference genes used in real-time RT-qPCR

机译：间充质基质细胞的3D封装和炎症许可改变实时RT-QPCR中使用的常见参考基因的表达

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摘要

Human mesenchymal stromal cells (hMSCs) hold great promise in the treatment of inflammatory and immune diseases, due to their immunomodulatory capacity. Their therapeutic activity is often assessed measuring levels of expression of immunomodulatory genes such as indoleamine 2,3-dioxygenase 1 (IDO1) and real-time RT-qPCR is most predominantly the method of choice due to its high sensitivity and relative simplicity. Currently, multiple strategies are explored to promote hMSC-mediated immunomodulation, overlooking the effects they pose in the expression of genes commonly used as internal calibrators in real-time RT-qPCR analyses. However, variations in their expression could introduce significant errors in the evaluation of the therapeutic potential of hMSCs. This work investigates, for the first time, how some of these strategies - 3D encapsulation, the mechanical properties of the 3D matrix and inflammatory licensing - influence the expression of common reference genes in hMSCs. Both 3D encapsulation and inflammatory licensing alter significantly the expression of beta-actin (ACTB) and Ubiquitin C (UBC), respectively. Using them as normalization factors leads to an erroneous assessment of IDO1 mRNA levels, therefore resulting in over or underestimation of the therapeutic potential of hMSCs. In contrast, the range of mechanical properties of the matrix encapsulating the cells did not significantly affect the expression of any of the reference genes studied. Moreover, we identify RPS13 and RPL30 as reference genes of choice under these particular experimental conditions. These results demonstrate the vital importance of validating the expression of reference genes to correctly assess the therapeutic potential of hMSCs by real-time RT-qPCR.
机译：人的间充质基质细胞（HMSCs）由于其免疫调节能力为治疗炎症和免疫疾病的治疗具有巨大的希望。它们的治疗活性通常评估免疫调节基因如吲哚胺2,3-二恶氧酶1（IDO1）的表达水平，并且实时RT-QPCR最主要是由于其高灵敏度和相对简单的选择方法。目前，探讨了多种策略来促进HMSC介导的免疫调节，忽略了它们在实时RT-QPCR分析中常用作为内部校准器的基因表达中的姿势。然而，其表达的变化可以在评估HMSCs的治疗潜力时引入重大误差。这项工作首次调查了这些策略 - 3D封装，3D基质和炎症许可的力学性能 - 影响HMSCs中共同参考基因的表达。 3D封装和炎症许可均显着改变β-肌动蛋白（ACTB）和泛素C（UBC）的表达。使用它们作为归一化因子导致IDO1 mRNA水平的错误评估，因此导致过度或低估HMSC的治疗潜力。相反，包封细胞的基质的力学性质的范围没有显着影响所研究的任何参考基因的表达。此外，在这些特定的实验条件下，我们将RPS13和RPL30鉴定为参考基因。这些结果表明，通过实时RT-QPCR验证参考基因表达以正确评估HMSCs的治疗潜力至关重要。

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  • 来源
    《Biomaterials Science》 |2020年第23期|共13页
  • 作者

    Gonzalez-Pujana Ainhoa; de Lazaro Irene; Vining Kyle H.; Santos-Vizcaino Edorta; Igartua Manoli; Maria Hernandez Rosa; Mooney David J.;

  • 作者单位

    Univ Basque Country UPV EHU Sch Pharm Lab Pharmaceut NanoBioCel Res Grp Vitoria Spain;

    Harvard Univ John A Paulson Sch Engn &

    Appl Sci Cambridge MA 02138 USA;

    Harvard Univ John A Paulson Sch Engn &

    Appl Sci Cambridge MA 02138 USA;

    Univ Basque Country UPV EHU Sch Pharm Lab Pharmaceut NanoBioCel Res Grp Vitoria Spain;

    Univ Basque Country UPV EHU Sch Pharm Lab Pharmaceut NanoBioCel Res Grp Vitoria Spain;

    Univ Basque Country UPV EHU Sch Pharm Lab Pharmaceut NanoBioCel Res Grp Vitoria Spain;

    Harvard Univ John A Paulson Sch Engn &

    Appl Sci Cambridge MA 02138 USA;

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  • 正文语种 eng
  • 中图分类 分子生物学;
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