ADM预处理间充质干细胞上调外泌体内lncRNAUCA1影响心肌细胞凋亡和治疗心肌病的机制研究

In order to maximize the effect of MSCs transplantation and use it in clinical, exploring the mechanism of MSCs transplantation becomes very important. Recent studies show that MSCs secrete exosome and exosome can replace cell transplantation and become an ideal mean to treat cardiovascular disease. Exosome can transfer lncRNA to adjacent cells and affect the function of these cells. However, the relationship amonge exosome, lncRNA and myocardium has not been reported. Our previous studies show that lncRNAUCA1 is involved in MSCs transplantation and ADM gene modification can improve the expression of lncRNAUCA1 in MSCs. Therefore, in this study we will use the methods of gene silencing, virus transfection, WB and rt-PCR, from cells to overall level, to prove that lncRNAUCA1 play an important role in mediating the antiapoptotic effect of exosome on cardiomyocyte and that exosome inhibit cell apoptosis by transferring lncRNAUCA1 to cardiomyocyte, and to search for the downstream target of lncRNAUCA1; Furthermore, we will confirme that ADM pretreatment can improve the expression of lncRNAUCA1 in exosome, and then improve the antiapoptotic effect of exosome. This study will provide a theoretical basis for exosome to treat heart failure and provide a new hope for the treatment of heart failure.

为提高MSCs移植的效果并应用于临床，探索MSCs移植的机制成为关键。新近研究表明MSCs分泌外泌体，并证实外泌体可代替细胞移植成为治疗心血管疾病的理想手段。外泌体可向邻近细胞内转移lncRNA进而影响细胞的功能。但是，外泌体、lncRNA和心肌之间的关系尚无报道。基于前期研究证实lncRNAUCA1参与MSCs移植，ADM基因修饰可提高MSCs内lncRNAUCA1的表达，本研究将利用基因沉默，病毒转染、WB及rt-PCR等技术，从细胞到整体水平证实lncRNAUCA1在外泌体抗心肌细胞凋亡中起重要作用，明确外泌体是将lncRNAUCA1转移到心肌细胞内从而抑制心肌细胞凋亡，同时寻找lncRNAUCA1的下游靶点；并证实ADM预处理可以提高外泌体内lncRNAUCA1的表达，进而提高外泌体抗心肌细胞凋亡。本研究将为外泌体移植治疗心力衰竭提供理论依据，也为心力衰竭的治疗提供一个新的希望。

近年来，外泌体移植已经成为心血管疾病治疗的一个理想手段。为了更好地使用外泌体治疗心血管疾病，如何提高外泌体的作用并明确外泌体治疗心血管疾病的机制是需要解决的关键问题。本研究主要是观察骨髓间充质干细胞（Bone mesenchymal stem cells, BMSCs）分泌的外泌体（Exo-BM）对心肌细胞凋亡的影响，同时观察肾上腺髓质素（Adrenomedullin，ADM）基因修饰BMSCs是否可以提高ADM-BMSCs分泌的外泌体(Exo-ADM）抗心肌细胞凋亡的能力，同时评估ADM基因修饰是否是通过Exo-ADM内某种microRNA的表达，提高Exo-ADM的抗心肌细胞凋亡能力。本研究结果表明，Exo-BM可通过进入心肌细胞内，减轻氯化镍诱导的心肌细胞的凋亡相关基因Caspase-3和细胞色素C的表达。而和Exo-BM相比，Exo-ADM可进一步抑制心肌细胞凋亡基因的表达；同时流式细胞仪检测也证实，和Exo-BM相比Exo-ADM可进一步抑制氯化镍诱导的心肌细胞凋亡。本研究进一步对Exo-BM和Exo-ADM进行基因检测及功能分析，最后选定miR-143和miR-26a作为可疑目的基因，并进一步对可疑目的基因进行功能验证。结果表明，miR-143与心肌细胞凋亡呈正相关，miR-26a与心肌细胞凋亡呈负相关。因此，通过本研究我们可以推断Exo-BM具有抗心肌细胞凋亡的能力，ADM基因修饰可通过提高BMSCs分泌外泌体内miR-26a的表达，进一步提高Exo-ADM的抗心肌细胞凋亡能力。