耐药脊柱结核特异性miRNA的筛选、鉴定及其作用机制研究

Spinal Tuberculosis (STB) is a chronic infectious disease caused by Mycobacterium tuberculosis (Mtb), which can lead to serious complications such as kyphosis. The rate of disability caused by improper treatment is extremely high. Due to the rapid growth of Mtb resistance, the treatment of drug-resistant spinal tuberculosis (M/XDR-STB) is extremely difficult, and there is no effective treatment in clinic.With the breakthroughs in gene sequencing and gene chip technology in recent years, it has been proved that microRNA could regulate human genes, and is a kind of nucleic acid substances with extensive functions. This study is aimed to obtain 5-15 differentially expressed microRNAs in plasma of MDR-STB, STB patients and healthy controls by high throughput sequencing and gene chip screening techniques. In this project, Targetscan and Go-KEGG analysis were used to screen 1-3 functional microRNAs and explore their value as potential new molecular markers for clinical diagnosis. MDR-TB Raw 264.7 cell line was established, by comparing its pharmacodynamics with isoniazid, the characteristics, structure-activity relationship and target of specific microRNAs in spinal tuberculosis were elucidated from three levels: cell function, signal transduction and gene conformation. The aim of this study to reveal its anti-drug-resistant role and mechanism of tuberculosis, expand new ways of treatment of drug-resistant tuberculosis.

脊柱结核（STB）是由结核分枝杆菌（Mtb）引起的慢性感染性疾病，可导致后凸畸形等严重并发症，治疗不当致残率极高。由于Mtb耐药性的快速增长，耐药脊柱结核（M/XDR-STB）的治疗极为棘手，临床缺乏有效的治疗途径。随着近几年基因测序、基因芯片技术的突破，证实MicroRNA类药效物质可调控人体基因，是一类作用广泛的核酸类物质。本项目采用高通量测序、基因芯片筛选等技术，确定MDR-STB、STB病人血浆、健康对照者血浆中5-15个差异表达miRNA，通过Targetscan、Go-KEGG分析，筛选1-3个功能性miRNA，探讨其作为临床诊断潜在新分子标志物的价值。建立MDR-TB Raw 264.7细胞株，通过与异烟肼进行药效对比，从细胞功能、信号转导和基因构象3个层次，阐明耐药脊柱结核特异性miRNA的作用特点、构效关系及作用靶标，揭示其抗耐药结核的作用及机制，拓展耐药结核治疗新途径。

脊柱结核（STB）是由结核分枝杆菌（Mtb）引起的慢性感染性疾病，可导致后凸畸形等严重并发症，治疗不当致残率极高。由于Mtb耐药性的快速增长，耐药脊柱结核（M/XDR-STB）的治疗极为棘手，临床缺乏有效的治疗途径。随着近几年基因测序、基因芯片技术的突破，证实MicroRNA类药效物质可调控人体基因，是一类作用广泛的核酸类物质。本项目采用高通量测序、基因芯片筛选等技术，确定MDR-STB、STB病人血浆、健康对照者血浆中5-15个差异表达miRNA，通过Targetscan、Go-KEGG分析，筛选功能性miRNA-133b，探讨其作为临床诊断潜在新分子标志物的价值。我们的研究结果显示：MDR-Mtb 的感染可以使脊柱结核病人体内miRNA 表达谱异常表达，其中 miRNA-133b在耐药脊柱结核病人体内显著低表达，细胞实验证实补充 miRNA-133b 对 MDR-Mtb 感染的 Raw 264.7 巨噬细胞具有显著保护作用，抑制MDR-Mtb 的生长,机制研究结果显示：MAML1过表达RAW264.7细胞系细胞活力显著降低，而转染miR-133b的细胞活力有显著的升高。相比对照组，在miR-133b瞬转和MAML1敲低的细胞系中，MAML1、IL-6、TNF-α、TGF-β的表达量均显著降低。实验揭示了micro-133b抗耐药结核的作用及机制，为后期拓展耐药结核治疗新途径提供一定的依据.