microRNA-29c在慢性移植物血管病中作用机制的研究

Chronic allograft vasculopathy (CAV) is always an important cause of the failure of organ transplantation. The endothelial cell （EC） and the vascular smooth muscle cell （VSMC） are the key effector cell types. In the previous study we found the expression level of miR-29c was significantly different between CAV samples in rat cardiac allograft transplant models compared to the isograft transplantation. The bioinformatic results about the target genes of miR-29c indicated the relation between this microRNA molecule and CAV. In this study we intend to adopt EC and VSMC as the research object in vitro. We expect to define the target genes of miR-29c in the primary EC and VSMC from the rat CAV model compared to the isograft through the QRT-PCR and Western blotting assay. We also intend to observe the difference in morphology, molecular markers and the biological function combined with the infection of lentivirus particles, which were packaged with the pMIF-cGFP-Zeo vector or the anti-sense miRZip-29c vector. The routine laboratory tests, including the MTT assay, the wound healing test, the Masson staining method and the EC-VSMC dual culture will be employed to evaluate the EC and VSMC status after miR-29c is dysregulated. The target genes should be verified through the dual luciferase reporter assay. The experiment in vivo will be carried out in the rat abdominal aorta allograft transplantation model, which reflects the pivotal role of miR-29c in the process of CAV. We propose that an optical miRNA-imaging strategy, based on molecular imaging, can be used as a miRNA imaging detector to monitor various miRNA levels, by using different receptors simultaneously. We anticipate this series of experiments will reveal the new molecular mechanism of CAV and serve as a foundation for the clinical development of therapeutic target.

CAV是器官移植失败的重要原因，EC和VSMC是CAV的重要效应细胞。我们在实验中发现miR-29c在发生CAV的大鼠心脏移植物中表达水平降低，生物信息学方法预测的靶基因中包括多个与CAV相关的分子。本研究拟构建慢病毒载体在大鼠动脉中原代分离的EC和VSMC中过表达或抑制表达miR-29c，研究细胞的形态、表型和功能的改变，并从发生CAV的动物模型中经磁珠分选得到EC和VSMC，在mRNA和蛋白水平研究预测的miR-29的靶基因的变化趋势及其与miR-29c变化的相关性，研究miR-29c这两种细胞中的调控作用；结合生物信息学的分析结果，构建双荧光素酶报告载体验证miR-29c的靶基因。在动物模型中高调表达miR-29c，验证其对CAV进程的影响。并进一步利用活体成像系统跟踪miR-29c在CAV进程中的转录和成熟的变化，本研究将有利于深入理解CAV的分子机制，并为临床诊疗方案提供新思路。

本研究实验结果主要包括两大部分。第一部分使用荧光素酶活性测定实验和实时荧光定量PCR等方法研究cx3cl1、cx3cr1、pdgfb和pdgfrb等可能与排斥反应发生和进展相关的基因，认为cx3cl1和pdgfrb等基因的3’非翻译区存在miR-29的调控序列，在转录后水平受miR-29的表达调控。研究miR-29的靶基因的表达变化是对其生物学作用研究的重要方面。第二部分是本研究的重点内容，构建miR-29的成像载体。传统的microRNA成像方式一般选择在一种荧光素酶末端加入microRNA的识别序列，通过与另一种内参荧光素酶的差值来使microRNA成像。在这种方法中，microRNA与携带其识别位点的荧光素酶的表达量成反比。由于microRNA的抑制作用并不完全，加之内参荧光素酶的存在，导致背景难以去除。此外，这种方法由于需要同时表达两种荧光素酶，给成像操作也带来了一定的复杂性。本研究利用Tet可诱导系统，在抑制性Tet分子（TetR）的末端加入microRNA的识别序列，通过消除TetR对其作用元件TetO下游荧光素酶的抑制作用，从而实现microRNA的成像。经过这种方法学的改进，只需要使用一种荧光素酶，且荧光素酶的表达与microRNA的表达是正相关的，从而克服了以往成像方法的缺陷，使microRNA的成像更为准确、方便。本研究用分子生物学方法构建了pcDNA4-TO-LUC报告质粒，包含可插入序列的抑制质粒pcDNA6-TR-TS，以及插入了miR-29种子序列或其对照序列的TetR表达质粒，共转染进入细胞系后，用荧光素酶的表达量成功指示了miR-29 mimics的表达变化。这将是我们研究正常和病理状态下miR-29在体内表达变化的重要工具。