超短程化疗治疗脊柱结核的基因表达谱评价的前瞻性对比研究

Shortening the course of treatment and improving the efficacy is a hot research topic in the treatment of spinal tuberculosis in recent years. Over the past decade, the course of an average of 4.5 months of ultra-short-course chemotherapy and thorough debridement in the treatment of spinal tuberculosis clinical research in our department, the results confirmed that the ultra-short-course chemotherapy is entirely feasible. But now at home and abroad is still a lack of modern bacteriology evaluation method of scientific evaluation of the molecular mechanism of the ultra-short-course chemotherapy and its efficacy. However, changes in gene expression in the detection of host peripheral blood mononuclear cells before and after treatment, you can quickly complete and unbiased show the efficacy of bacterial infection.In this study, we are the first to adopt whole-genome wide expression profiling technology, select ultra-short-course and standard chemotherapy treatment of spinal tuberculosis patients to do the following reaserch by prospective comparative study：monitor their treatment of different stages of peripheral blood mononuclear cell RNA expression profiles change, elect to participate in the body's immune mechanisms of mycobacterium tuberculosis differentially expressed genes and functional analysis, and express protein product of the critical gene expression in the rabbit spinal tuberculosis model validation experiments. In order to reveal the different stages of treatment of ultra-short-course chemotherapy of spinal tuberculosis bacteria elimination of variation；evaluation methods from molecular level to explore the efficacy and theoretical basis of the ultra-short-course chemotherapy treatment of spinal tuberculosis.

缩短疗程、提高疗效是近年脊柱结核病防治研究的热门课题。近十余年来，本部门进行了疗程平均4.5个月的超短程化疗并彻底病灶清除术治疗脊柱结核的临床研究，结果证实超短程化疗是完全可行的。但是目前国内外尚缺乏现代细菌学的评价方法对超短程化疗的分子机制及其疗效进行科学评价。然而，通过检测宿主外周血单核细胞在治疗前后基因量表达的变化，可以快速、完整、无偏差地显示对细菌感染干预的疗效。本研究首次采用基因表达谱芯片技术，对选择超短程化疗及标准化疗治疗的脊柱结核患者进行前瞻性对比研究：检测不同治疗阶段患者外周血单核细胞RNA表达谱变化；筛选出参与机体对结核杆菌免疫机制的差异基因并进行功能分析；将关键基因表达出的蛋白产物在兔脊柱结核模型中进行免疫机制验证实验。从而揭示超短程化疗脊柱结核不同治疗阶段的细菌消长变化规律；从分子水平探讨超短程化疗治疗脊柱结核的理论依据与疗效评价方法。

缩短疗程、提高疗效是近年脊柱结核病防治研究的热门课题。本部门临床研究结果证实超短程化疗治疗脊柱结核是完全可行的。但是目前国内外尚缺乏现代细菌学的评价方法对超短程化疗的分子机制及其疗效进行科学评价。本研究采用基因表达谱芯片技术，对选择超短程化疗治疗的脊柱结核患者进行前瞻性对比研究：探讨超短程化疗前后脊柱结核患者临床疗效与外周血mRNA基因表达谱差异基因变化的关系；研究KLF4调控的TLR-2/p38MAPK/NF-κB信号通路在脊柱结核发病机制中的作用。. 本研究结果显示如下：.1. 临床结果显示未治疗组（A组）结核病灶活动；超短程化疗结束组（B组）、随访一年组（C组）两组结核病灶治愈。SAM分析结果显示，A、B组之间差异表达基因共942条，上调基因936条、下调基因6条； A、C组之间差异表达基因276条，上调基因256条、下调基因20条；B、C组之间无显著差异表达基因。MAS分析显示差异基因主要参与炎症反应及宿主的免疫调节机制的过程。A组较B、C两组TNF-α、IFN-γ、IL-12、IL-8表达水平明显升高，这与NF-κB的表达水平相一致。.2. 芯片结果提示脊柱结核组和正常对照组差异表达基因68条，上调基因16条，下调基因52条，其中KLF4基因在脊柱结核患者外周血中表达最为显著，升高2.89倍。RT-PCR结果显示脊柱结核组外周血KLF4、TLR-2、p38、NF-κB基因在mRNA水平均高表达，IFN-γ、TNF-α、IL-1表达亦升高，同时KLF4、TLR-2、p-p38、NF-κB蛋白表达水平增加，但p38蛋白水平却无明显变化。.3. 在100 ng/mL LPS作用后的巨噬细胞中TLR-2、p-p38、NF-κB、KLF4在mRNA水平和蛋白表达水平明显增加，IFN-γ、TNF-α及IL-1的表达也明显升高。. 本研究结果表明超短程化疗治疗脊柱结核的临床疗效与外周血mRNA差异基因、炎性细胞因子及核转录因子κB蛋白表达水平一致。外周血mRNA表达谱芯片可用于评估在彻底病灶清除术基础上采用超短程化疗治疗的脊柱结核患者的临床疗效。外周血Krüppel样因子4可能通过诱导单核细胞向巨噬细胞分化而参与脊柱结核的发病，其可能通过TLR-2/p38MAPK/NF-κB途径参与脊柱结核炎症反应的发生。