脊柱结核血清、尿液中特异循环miRNA标记物的筛选验证及其在成骨细胞分化中的调控机制研究

The role of the diagnosis for spinal tuberculosis by clinical manifestations , radiographic changes and laboratory tests are limited, and there is no specific molecular biology detection.Based on global miRNA research discovery in recent years, it is necessary to study the circulating miRNAs differentially expressed in serum and urine which can be as a specific biological markers to diagnosis spinal tuberculosis. In our previous study, we have screened out a miRNA named hsa-miR-30c-5p which is found in the serum and it’s also the most close to spinal tuberculosis , and we have successfully completed the separation and extraction of circulating miRNA in the urine. This study intends to plans to complete the following goals : the screening, clinical validation，and regulatory pathway targeted verification about differentially circulating miRNA in serum and urine of spinal tuberculosis’; targeted verification about the hsa-miR-30c-5p; analysis the sensitivity and specificity of circulating miRNA markers in serum and urine and also the correlation Through the above study, we are trying to establish the associated circulating miRNA in serum and / or urine which can be used as biomarkers to diagnosis spinal tuberculosis ,and to make clear about the mechanism of hsa-miR-30-5p’sregulatory in osteoblast cell differentiation .So as to provide a new method for accurate diagnosis of spinal tuberculosis, and provide a theoretical basis for the treatment and evaluation of therapeutic effect.

脊柱结核病的临床表现、影像学改变、实验室检查的诊断作用有限，尚无具有特异性的分子生物学检测方法。基于近年全球miRNA研究的重大发现，很有必要进行血清、尿液中差异表达的循环miRNAs作为诊断脊柱结核病特异性生物标记物的研究。我们前期研究已从血清中筛选出了与脊柱结核最为贴近的差异表达的hsa-miR-30c-5p;并在尿液中成功分离、提取了循环miRNA。本研究拟计划完成：脊柱结核血清、尿液中差异循环miRNA的筛选、临床验证及靶向调控通路验证；hsa-miR-30c-5p的靶向性验证；血清、尿液中循环miRNA标记物灵敏度、特异度及二者的相关性分析。经上述研究，力图确立血清或/和尿液中相关循环miRNA可作为脊柱结核诊断的特异性生物标记物，明确hsa-miR-30-5p在成骨细胞分化中的调控机制。从而为脊柱结核病的精准诊断提供一个新方法，并为治疗、疗效评价等方面的研究提供理论基础。

脊柱结核病的临床表现、影像学改变、实验室检查的诊断作用有限，尚无具有特异性的分子生物学检测方法。在本研究中首先使用 miRNA基因芯片对脊柱结核和正常健康人的血浆miRNA以及尿液外泌体miRNA进行高通量筛选。然后使用RT-qPCR技术对部分芯片筛选结果进行了三个阶段四个队列的大样本验证，最终得到对于脊柱结核具有诊断价值的一组miRNA。以上述差异miRNA为切入点，对结核杆菌侵染成骨细胞后通过hsa-miR-543靶向调控RANKL表达的机制进行了初步研究。研究发现：联合脊柱结核患者血浆hsa-miR-506-3p，hsa-miR-543、hsa-miR-195-5p作为脊柱结核的诊断模型，其敏感性=80.5％，特异性=80.0%；联合脊柱结核患者尿液外泌体hsa-let-7f-5p、hsa-miR-4768-3p、hsa-miR-4437、hsa-miR-381-5p作为脊柱结核的诊断模型，其敏感性=91.1％，特异性=84.3%；H37Ra pMV261-GFP荧光菌株转染入成骨细胞后会降低hsa-miR-543的表达从而促进RANKL蛋白的表达。本研究最终各筛选出一组脊柱结核血浆miRNA和尿液外泌体miRNA诊断标记物，两者均具备成为脊柱结核的特异性诊断标记物的潜力。并初步明确了结核杆菌侵入成骨细胞后，通过降低hsa-miR-543的表达，从而促进RANKL表达以促进破骨细胞活化的机制。