用信使核糖核酸（mRNA）改造犬脂肪组织来源的间充质干细胞，使其过表达TSG-6并增强犬巨噬细胞的抗炎作用。
Canine adipose tissue-derived MSCs engineered with mRNA to overexpress TSG-6 and enhance the anti-inflammatory effects in canine macrophages.
作者信息
Yun Ga-Hee, Park Su-Min, Lim Ga-Hyun, Seo Kyoung-Won, Youn Hwa-Young
机构信息
Laboratory of Veterinary Internal Medicine, Department of Clinical Veterinary Science, College of Veterinary Medicine, Seoul National University, Seoul, Republic of Korea.
出版信息
Front Vet Sci. 2023 Apr 6;10:1134185. doi: 10.3389/fvets.2023.1134185. eCollection 2023.
BACKGROUND
Mesenchymal stem cells (MSCs) are useful agents in the treatment of various inflammatory diseases. The immunomodulatory effects of MSCs are largely related to their secretory properties. mRNA engineering emerged as a safe alternative to enhance the secretory function of MSCs. Optimization of the untranslated region (UTR) sequence is important for enhancing the translational efficiency of exogenous mRNAs. However, research on the optimization of UTR in canine MSCs has not yet been conducted.
OBJECTIVES
We aimed to identify the UTR sequence related to the expression efficiency of transcription (IVT) mRNA in canine MSCs and investigate whether mRNA-engineered MSCs that overexpress TSG-6 exhibit enhanced anti-inflammatory effects.
METHODS
Canine adipose tissue-derived (cAT)-MSCs were transfected with mRNA with three different UTRs: , and . The translation efficacy of each mRNA was evaluated using relative fluorescence. mRNA was produced with the UTR optimized according to relative fluorescence results. cAT-MSCs were transfected with mRNA (MSC), and TSG-6 expression was analyzed using real-time quantitative PCR, ELISA, and western blotting. To evaluate the anti-inflammatory effects of MSCs, DH82 cells were co-cultured with MSCs or treated with dexamethasone, and changes in the expression of inflammatory cytokines were analyzed using qPCR.
RESULTS
The highest fluorescence level was observed in the UTR at 24 h post-transfection. mRNA transfection yielded high levels of TSG-6 in the cAT-MSCs. In DH82 cells co-cultured with MSCs, the expression of inflammatory cytokines decreased compared to that in co-culturing with naïve MSCs and dexamethasone treatment.
CONCLUSIONS
Optimization of the UTR improved the translation efficiency of IVT mRNA in canine MSCs. cAT-MSCs engineered with mRNA effectively enhanced the anti-inflammatory effects of the MSCs when co-cultured with LPS-activated DH82 cells.
背景
间充质干细胞（MSCs）是治疗各种炎症性疾病的有效药物。MSCs的免疫调节作用很大程度上与其分泌特性有关。mRNA工程作为一种安全的替代方法出现，用于增强MSCs的分泌功能。非翻译区（UTR）序列的优化对于提高外源mRNA的翻译效率很重要。然而，尚未对犬MSCs中UTR的优化进行研究。
目的
我们旨在鉴定与犬MSCs中转录（IVT）mRNA表达效率相关的UTR序列，并研究过表达TSG-6的mRNA工程化MSCs是否表现出增强的抗炎作用。
方法
用三种不同UTR的mRNA转染犬脂肪组织来源（cAT）-MSCs： 、 和 。使用相对荧光评估每种mRNA的翻译效率。根据相对荧光结果优化UTR来生产mRNA。用mRNA（MSC）转染cAT-MSCs，并使用实时定量PCR、ELISA和蛋白质印迹分析TSG-6表达。为了评估MSCs的抗炎作用，将DH82细胞与MSCs共培养或用地塞米松处理，并使用qPCR分析炎症细胞因子表达的变化。
结果
转染后24小时在UTR中观察到最高荧光水平。mRNA转染在cAT-MSCs中产生高水平的TSG-6。在与MSCs共培养的DH82细胞中，与未处理的MSCs共培养和地塞米松处理相比，炎症细胞因子的表达降低。
结论
UTR的优化提高了犬MSCs中IVT mRNA的翻译效率。用mRNA工程化的cAT-MSCs在与LPS激活的DH82细胞共培养时有效地增强了MSCs的抗炎作用。
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