“核HO-1”调控miRNA-125a-5p影响血脊髓屏障结构和功能的机制研究

Spinal cord injury (SCI) is one kind of serious injuries in Central Nervous System, accompanied with the disruption of blood-spinal cord barrier (BSCB). The study demonstrated that the disruption of BSCB is involved in the pathophysiologyic process of SCI. And the repair of BSCB will lead to a significant neuroprotective effect. BSCB is a metabolism barrier between the spinal cord tissue and blood circulation, which strictly keep the microenvironment of spinal cord. Heme oxygenase 1 (HO-1) is a stress inducible gene, played an important role in heme metabolism. Recently, the function of "nuclear translocation" draw the attention of people: the "nuclear HO-1" protein itself might have an important role in the regulation of intracellular signal transduction. And HO-1 is not a traditional transcription factor, therefore, its regulation on gene expression is likely to play an indirect role. Recently, microRNAs (miRNA) have emerged as a critical class of regulators of gene expression. miRNA are endogenous small 21-23 nucleotide nonocoding RNAs that possess remarkable evolutionary conservation.It has been proved that, miRNA is widely present in eukaryotic cells, is one of the largest gene families, account for about 1% of the whole genome, plays an important role in the regulation of gene expression and biological growth process. Disorders of any miRNA will result in the change of the function of cells. Therapeutic application of microRNAs, such as miRNA-125a-5p, potentially could re-establish normal function of the brain vasculature in endothelial cell-based neurological diseases. At present, researchers began to pay attention to the effect of HO-1 on miRNA function, and found the HO-1 could regulate the expression of miRNA by binding to specific miRNA promoter or a specific sequence and then realize the protective role on cells. To search for miR-125a-5p which may be involved in the regulation of BSCB permeability induced by SCI. After the plasmid carried HO-1C Δ 23 was transfected into vascular endothelial cells, miRNA-125a-5p expression was markedly up-regulated and BSCB permeability induced by hypoxia and ROS was significantly reduced. Therefore, we persume that "nuclear HO-1" is an important target for the regulation of BSCB through miRNA-125a-5p.The first aim of this study is to investigate the effects of "nuclear HO-1" on structure and function of BSCB in vitro under different injury conditions. The second aim of this project are to investigate the role of miRNA-125a-5p in regulating the BSCB induced by "nuclear HO-1". Finally, we investigated that the effect o exogenous recombinant adenovirus of "nuclear HO-1" (Ad-HO-1C Δ 23) on BSCB permeability and the nervous function after SCI. This study will clarify that the role and mechanism of "nuclear HO-1" in regulating the BSCB, providing the theoretical and experimental evidences to fully understand the related mechanisms, and create a new strategy for drug development in SCI.

脊髓损伤是一种严重的中枢神经系统损伤，伴有血脊髓屏障(blood-spinal cord barrier,BSCB)的破坏。研究证实"核HO-1"可参与基因调控，维持血管内皮细胞正常功能。本课题组将携带HO-1CΔ23（"核HO-1")的质粒转染入血管内皮细胞中，证实转染"核HO-1"的血管内皮细胞，miRNA-125a-5p表达显著上调，且显著降低缺氧或ROS诱导的BSCB的通透性。因此，我们推测"核HO-1"是调控BSCB通透性的重要靶点。本项目首先研究"核HO-1"对BSCB结构和功能的影响；进一步研究miRNA-125a-5p在"核HO-1"调节BSCB中的作用和机制；最后研究外源性给予表达"核HO-1"的重组腺病毒，对脊髓损伤后BSCB通透性和神经功能的影响。本项目首次揭示了"核HO-1"改善脊髓损伤后BSCB的功能和调控机制，丰富了脊髓损伤修复理论，为脊髓损伤的治疗开创新靶点。

“核HO-1”被证实可参与基因调控，维持血管内皮细胞正常功能，本项目首先在细胞水平上建立体外血脊髓屏障模型和缺氧模型，进一步通过病毒介导完成了体外“核HO-1”的导入。我们将携带HO-1CΔ 23（“核HO-1”)的质粒转染入血管内皮细胞中，明确“核HO-1”能够降低BSCB的通透性。通过电泳泳动迁移分析技术我们证实了转染“核HO-1”的血管内皮细胞能够显著提高miRNA-125a-5p的表达。对紧密连接蛋白ZO-1等蛋白的研究进一步证实“核HO-1”可以调节BSCB的通透性。因此可以表明“核HO-1”可以通过miRNA-125a-5p改善脊髓损伤后BSCB 的功能和调控机制，维持血管内皮细胞正常功能，同时对脊髓损伤后神经元细胞的保护有正向作用。通过大鼠建立脊髓损伤模型，外源性给予表达“核HO-1”的重组腺病毒，通过Western blots和免疫荧光技术检测不同时间点紧密连接相关蛋白的表达水平和分布变化。本研究通过脊髓水肿程度、脊髓血流以及BBB评分证实外源性给予表达“核HO-1”能够改善神经功能，从而减缓继发性脊髓损伤。综上，本研究结果系统阐述了“核HO-1”通过miRNA-125a-5p可以改善脊髓损伤后BSCB 的功能和调控。同时阐述了“核HO-1”对脊髓损伤后神经元细胞的保护有正向作用。课题也丰富了脊髓损伤修复理论，为脊髓损伤的治疗开创新靶点。项目资助发表SCI 5篇，核心论文3篇。培养硕士研究生9名，其中6人已经获得硕士学位，3人在读。