Regulation of renal and bone marrow injury by extracellular vesicle non-coding RN

描述(由申请人提供)：最近的研究表明，细胞衍生的囊泡可以通过转移RNA物种来改变单独目标细胞的基因组表型。进一步的研究表明，间充质干细胞衍生的囊泡(MSC-DV)可以通过转移microRNA来介导损伤的肾和骨髓组织的修复。一个关键的需要是确定MSC-DV是否可以用来逆转如此痛苦的人类的肾脏或骨髓损害。本研究的目的是确定分离和鉴定携带“修复”microRNA的MSC-DV的最佳方法，MSC-DV可用于甘油暴露或缺血/再灌注损伤的肾组织或辐射损伤的骨髓组织(干细胞)。然后，将通过深度测序确定“修复”MSC-DV中的microRNA，并通过将其输送到受损的肾脏和骨髓细胞系模型并恢复增殖和减少凋亡的能力来确定特定的“修复”microRNA。这将通过脂质体感染受损的细胞系并分析增殖和凋亡来实现。我们还将描述用特定的“治愈”微RNA装载“治愈”MSCDV的最佳方法(S)。预计这些研究将产生以下预期结果：1)了解微型RNA在组织修复中的作用，2.对负责细胞命运改变的特定微RNA或微RNA子集的定义和3.)这项研究为MSC囊泡microRNA的输送提供了一种途径，以修复受损的肾脏或骨髓组织。这是提案的第一阶段。在第二阶段，在确定了最佳包装在MCS-DV中的“愈合”microRNA以输送到受伤的骨髓或肾组织后，我们接下来需要优化组织修复的输送时间表，评估早期和晚期毒性，以及注入剂的分布。此外，我们还需要评估富含microRNA的MSC-d囊泡在储存过程中“愈合”表型的稳定性。最佳的愈合水平将在时间进程实验中评估，在我们的模型中，不同的输液时间和频率被评估为愈合效果。在这些研究过程中，我们还将确定输液动物的早期和晚期毒性。还将评估整个动物器官的组织学和存活率。 接下来，我们将跟踪最佳输液计划后的组织分布，并确定不同条件下MSC-DV随时间的稳定性。第一阶段和第二阶段概述的所有研究都是独一无二的，具有创新性。在MSC-DV中运送microRNA用于治疗组织损伤的目的是为以前从未尝试过的不同肾脏和骨髓疾病的治疗干预准备基础。

DESCRIPTION (provided by applicant): Recent studies indicate that cellular derived vesicles can alter the genomic phenotype of separate target cells by transfer of RNA species. Further work has indicated that mesenchymal stem cell-derived vesicles (MSC-dv) can mediate healing of injured renal and marrow tissue by transfer of microRNA. A critical need is to determine whether MSC-dv can be utilized to reverse kidney or marrow damage in humans so afflicted. The present proposal is to define the optimum approach for separating and characterizing MSC-dv carrying "healing" microRNA to renal tissue injured by glycerol exposure or by ischemia/reperfusion or marrow tissue (stem cells) injured by irradiation. The microRNA in "healing" MSC-dv will then be determined by deep sequencing and the specific "healing" microRNA determined by its capacity to be delivered to injured renal and marrow cell line models and restore proliferation and reduce apoptosis. This will be carried out by lipofecting injured cell lines and analyzing proliferation and apoptosis. We will also characterize optimal approaches to loading "healing" MSC-dv with specific "healing" microRNA(s). It is anticipated that these studies will yield the following expected outcomes: 1.) An understanding of the role of micro RNAs in tissue restoration, 2.) a definition of the specific micro RNA or subset of microRNAs responsible for cell fate change and 3.) the development an approach for the delivery of MSC vesicle microRNA to restore injured renal or marrow tissue. This constitutes Stage 1 of the proposal. In Stage 2, after the determination of a "healing" microRNA packaged optimally in MCS-dv for delivery to injured marrow or renal tissue, we next need to optimize delivery schedule for tissue repair, evaluate early and late toxicity, and distribution of the infued agent. In addition, we need to evaluate the stability of "healing" phenotype of the microRNA rich MSC-d vesicles with storage. Optimal healing levels will be assessed in time course experiments where different times and frequencies of infusion are evaluated as to healing effects in our models. During the course of these studies we will also determine early and late toxicities in the infused animals. Total animal organ histology and survival will also be assessed. Next we will track tissue distribution after an optimal infusion schedule and determine the stability of MSC-dv over time under different conditions. The totality of the studies outlined in Stage 1 and 2 are unique and innovative. The delivery of microRNA in MSC-dv to heal tissue injuries is designed to prepare the basis for therapeutic interventions in different renal and marrow diseases which have not been previously attempted.