急性心肌梗死患者培养的人间充质干细胞(MSCs)对无细胞DNA (cfDNA)的早期和晚期反应

IF 3.3 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY
Elena M Malinovskaya, Natalia N Veiko, Elisaveta S Ershova, Larisa V Kameneva, Marina S Konkova, Svetlana V Kostyuk
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引用次数: 0

摘要

背景:急性心肌梗死(AMI)伴有心脏组织损伤和部分细胞死亡。干细胞定位于患处,有助于组织修复。既往研究表明AMI患者血液中游离DNA (cfDNA) (AMI -cfDNA)浓度显著升高,且AMI -cfDNA组成中含有富含gc的氧化DNA片段。因此,ami-cfDNA在体外对各种类型的分化人类细胞表现出生物活性。潜在地,ami-cfDNA可以影响干细胞分化的功能活性和方向。为了验证这一假设,我们在体外研究了从AMI患者血液中分离的AMI - cfdna片段对人脂肪组织间充质干细胞(MSCs)的影响。材料和方法:采用干细胞表面标记物对MSC细胞系进行表征。从7例AMI患者和10例健康供者的血浆中分离出AMI - cfdna和对照(hc-cfDNA)。分析MSCs对cfDNA作用的早期(0.5-3小时)和晚期(1-3周)反应。采用定量(实时)聚合酶链式反应(qPCR)、流式细胞术、荧光显微镜、荧光原位杂交(FISH)等检测方法,检测活性氧水平、多个基因(NOX4、NRF2、BRCA1、BCL2、BAX、MYOD1、MYOG、MYF5、MRF4、RUNX2、SPP1、OCN、LPL、AP2)的表达水平、细胞核内双链DNA断裂水平以及细胞核内染色质空间组织的变化。结果:在细胞培养基中引入ami-cfDNA片段可刺激间充质干细胞快速、短暂地诱导氧化应激(早期反应)。氧化应激刺激染色质的空间重组以产生适应性反应(AR)。适应性反应包括抗氧化和抗凋亡反应以及修复基因的激活。与hc-cfDNA不同,ami-cfDNA片段在长时间暴露(晚期反应)下刺激MSCs的肌源性分化。结论:ami-cfDNA通过诱导明显的适应性细胞反应增加了模型系统中MSCs的存活。长时间暴露于ami-cfDNA中会引起MSCs的肌源性分化。在体内AMI引起的急性应激状态下,AMI - cfdna可能积极影响受损心肌的修复。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Early and Late Responses of Cultured Human Mesenchymal Stem Cells (MSCs) to Cell-free DNA (cfDNA) in Patients With Acute Myocardial Infarction.

Background: Acute myocardial infarction (AMI) is accompanied by damage to heart tissues and some cell death. Stem cells are localized in the affected area and contribute to tissue repair. Studies have previously shown that the concentration of cell-free DNA (cfDNA) in the blood (ami-cfDNA) increases significantly in patients with AMI, and GC-rich and oxidized DNA fragments accumulate in the composition of ami-cfDNA. As a result, ami-cfDNA exhibits biological activity in vitro against various types of differentiated human cells. Potentially, ami-cfDNA can influence the functional activity and direction of stem cell differentiation. To verify this assumption, we investigated the effect of ami-cfDNA fragments isolated from the blood of patients with AMI on human adipose tissue mesenchymal stem cells (MSCs) in vitro.

Materials and methods: The MSC line was used and characterized by stem cell surface markers. Ami-cfDNA and control (hc-cfDNA) samples were isolated from the blood plasma of seven AMI patients and ten healthy donors. The early (0.5-3 hours) and late (1-3 weeks) responses of MSCs to cfDNA action were analyzed. The level of reactive oxygen species, the expression level of numerous genes (NOX4, NRF2, BRCA1, BCL2, BAX, MYOD1, MYOG, MYF5, MRF4, RUNX2, SPP1, OCN, LPL, AP2), the level of double-stranded DNA breaks in nuclei, and changes in the spatial organization of the chromatin in the nucleus were determined using the quantitative (real-time) polymerase chain reaction (qPCR), flow cytometry, fluorescence microscopy, fluorescent in situ hybridization (FISH) assays.

Results: Introducing ami-cfDNA fragments into the cell culture medium stimulates rapid and transient induction of oxidative stress in MSCs (early response). Oxidative stress stimulates the spatial reorganization of chromatin to develop an adaptive response (AR). The adaptive response includes an antioxidant and anti-apoptotic response and activation of repair genes. The ami-cfDNA fragments, unlike hc-cfDNA, stimulate the myogenic differentiation of MSCs under prolonged exposure (late response).

Conclusions: The ami-cfDNA increases the survival of MSCs in the model system by inducing a pronounced adaptive cellular response. Prolonged exposure to ami-cfDNA provokes myogenic differentiation of MSCs. Under acute stress conditions caused by AMI in the body, ami-cfDNA may positively affect the restoration of damaged heart muscle.

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