摘要
[目的]探讨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。[方法]实验组为经手术及病理证实的8例脊柱结核患者,对照组为5例健康人群。收集并对比分析A、B两组的临床表现、血常规、ESR、C-RP。采集外周静脉血10 ml,提取总RNA,利用表达谱基因芯片检测A、B两组外周血白细胞差异表达的mRNA基因,筛选出在脊柱结核患者中高表达的KLF4基因。初步验证采用以下三种方法:应用RT-PCR检测外周血中KLF4、TLR-2、p38、NF-κB基因在mRNA水平的表达;应用ELISA法对IFN-r、TNF-α、IL-1进行检测;应用Western Blot法检测外周血白细胞中KLF4、TLR-2、磷酸化p38(p-p38)、p38、NF-κB蛋白的表达。[结果]临床结果显示,相对于正常对照组,脊柱结核组ESR、CRP、中性粒细胞相对值升高,淋巴细胞绝对值、淋巴细胞相对值及血红蛋白值降低。芯片结果提示脊柱结核患者与正常对照组之间显著差异表达基因共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差异表达最为显著,升高2.89倍。RT-PCR结果显示脊柱结核患者较正常对照组外周血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蛋白水平无明显变化。[结论]病灶活动期脊柱结核患者外周血KLF4基因表达升高可能通过TLR-2/p38MAPK/NF-κB途径参与脊柱结核炎症反应。
[Objective] To evaluate the function of the Krüppel-like factor 4（ KLF4） genein monocytic differentiation and its regulation in Toll-like receptor-2（ TLR-2） / p38mitogen-activated protein kinase（ MAPK） / nuclear factorκB（ NF-κB） signaling pathway in the pathogenesis of spinal tuberculosis. [Methods] The study group was composed of 8 spinal tuberculosis（ TB） patients which had been confirmed by surgery and pathology. The control group was made up of 5 healthy people. All the clinical data,including blood routine examination,erythrocyte sedimentation rate（ ESR）,C-reactive protein（ CRP） were collected and comparatively analyzed. 10 ml peripheral blood was collected for extracting of total RNA,the mRNA gene expression microarray was used to detect the gene changes in the peripheral blood samples,and KLF4 gene was screened in spinal tuberculosis patients with highest expression. Real-time PCR（ RT-PCR） was performed to confirm the changes in the mRNA expression of TLR-2,p38,NF-κB,and KLF4 genes. Interferon-gamma（ IFN-r）,tumor necrosis factor（ TNF-α）,and interleukin-1（ IL-1） were detected by enzyme-linked immunosorbent assay（ ELISA） method. Human monocytes（ THP-1） was induced to be macrophages by phorbol-12-myristate-13-acetate（ PMA）. The expression of mRNA and protein of TLR-2,p-p38,p38,NF-κB,and KLF4 in macrophages after lipopolysaccharide（ LPS） treated were detected. IFN-γ,TNF-α,and IL-1 were also tested using ELISA method. [Results] Clinical results showed that ESR,CRP,and the relative value of neutrophils increased, but absolute and relative value of lymphocytes and hemoglobin values decreased. The microarray results showed 68 genes with significant difference between spinal tuberculosis patients and normal control group,16 genes up-regulated, and 52 genes down-regulated. Among the genes,KLF4 gene was the most significant differentially up-regulated gene which was increased by 2. 89 times in spinal tuberculosis patients. RT-PCR showed that there was a significant difference in the high mRNA expression of TLR-2,p38,NF-κB,and KLF4 in spinal tuberculosis patients compared with those of control group. The expressions of TLR-2,p-p38,NF-κB,KLF4 significantly increased in the level of mRNA and protein. TNF-α,IFN-γ,and IL-1 expression levels also significantly increased after macrophages treated by100 ng / ml LPS. [Conclusion] KLF4 gene may cause inflammation by participating in TLR-2 / p38 MAPK / NF-κB signaling pathway. KLF4 genes participate in anti-tubercular reactions involving in spinal tuberculosis by regulating monocyte to macrophage differentiation.
出处
《中国矫形外科杂志》
CAS
CSCD
北大核心
2016年第7期636-642,共7页
Orthopedic Journal of China
基金
国家自然科学基金课题(编号:81260282)
宁夏自然科学基金课题(编号:NZ11195
NZ11275)
作者简介
作者简介：王骞，医学硕士，研究方向：脊柱结核，（电话）13709515798，（电子信箱）jetwangl@hotmail．com
通讯作者：（电话）0951-6743242，13629593280，（电子信箱）niun-ingkui@163．com
