| ObjectiveEvaluate the relationship between the clinical efficacy of ultra-short-course chem-otherapy(UCCT) for spinal tuberculosis and peripheral blood gene expressions befo-re and after UCCT with gene microarray technology, and study the function of the Krüppel-like factor 4(KLF4) gene in monocytic differentiation and its regulation in Toll-like receptor-2(TLR-2)/p38 mitogen-activated protein kinase(MAPK)/ nuclear factorκB(NF-κB) signaling pathway in the pathogenesis of spinal tuberculosis.Methods1. The study group was composed of 27 spinal TB patients who were admitted int-o the department of spinal surgery in the General Hospital of Ningxia Medical University, and the patients were treated by complete debridement and cured by UCCT, and also the patients were confirmed by surgery and pathology. Those patients were 13 males and 14 females,and aged between 17 to 72 years, and the mean age was 38.8±15.6 years. The 27 spinal TB patients were divided into three groups: Grou-p A(untreated group consisting of 8 cases), Group B(treatment-completed group of9 cases), and Group C(1-year follow-up group of 10 cases). All the patients were treated by the same chemotherapy of ultra-short-course chemotherapy(2SHRZ/2-4HRZ), and surgery programs were complete debridement and inter bo-dy graft fusion and internal fixation between the focal vertebras. The control group was made up of the peripheral blood samples from 5 non TB patients. Those patients were 3males and 2 females, and aged between 25 to 50 years, and the mean age was35.2±12.7 years.2. Collected all the patients’ peripheral blood 10 ml,extracted total RNA, used of the expression of Affymetrix U133 Plus 2.0 Array mRNA gene microarray to detect the gene changes in the peripheral blood samples. Significance analysis of microarrays(SAM) was employed to identify differentially expressed genes and followed by cluster analysis. The Molecule Annotation System(MAS) was used to identify the pathways and the functions of the differentially expressed genes. Reversetranscription-PCR(RT-PCR)was performed to confirm the changes in the expression of the target genes.3. Collected and comparative analyzed all the clinical data of subjects, and obser-ved the clinical efficacy of UCCT for spinal tuberculosis. Tumor necrosis factor(TNF-α), Interferon-gamma(IFN-γ), Interleukin-12(IL-12), Interleukin-8(IL-8), and NF-κB were detected in all the peripheral blood samples with different stages of treatment by enzyme-linked immunosorbent assay(ELISA) method,also detected the protein of NF-κB by Western blot method.4. Used of the expression of mRNA gene microarray to detect the gene changes between spinal tuberculosis patients and normal control group in the peripheral blood samples, and screened KLF4 gene in spinal tuberculosis patients with highest expression. Collected and comparative analyzed all the clinical data of subjects, blood routine examination, erythrocyte sedimentation rate(ESR), C-reactive protein(CRP),and film examination. RT-PCR was performed to confirm the changes in the mRNA expression of TLR-2, p38, NF-κB, and KLF4 genes between spinal tuberculosis patients and normal control group. IFN-γ, TNF-α, and Interleukin-1(IL-1) were detected by ELISA method. Detecting the expression of protein of KLF4, TLR-2, p–p38MAPK, p38 MAPK, and NF-κB in the patients’ peripheral blood. Human monocytes(THP-1) were induced to be macrophages by phorbol-12–myris-tate-13-acetate(PMA). Detecting the expression of m RNA and protein of TLR-2, p-p38,p38,NF-κB,and KLF4 in macrophages after lipopolysaccharide(LPS) treated.IFN-γ, TNF-α, and IL-1 were also detected.Results1. Clinical result showed the tuberculosis lesions in group A were still active; whi-le whether the clinical manifestation or lab and imaging exam all indicated the cure of tuberculosis lesions in both Group B and Group C, and there were no significant difference. The experimental result, among the 942 genes differentially expressed genes between Group A and Group B, 936 genes were unregulated and 6 were down regulated. Among the 276 differentially expressed genes between Group A and Group C, 256 genes were unregulated and 20 genes were down regulated. No differentiallyexpressed genes were identified between Group B and Group C. MAS analysis showed the functions of these differentially expressed genes were mainly related to the immune regulation of patients. RT-PCR indicated similar findings to that of the gene microarray assay. Group A compared with Group B and Group C showed that TNF-α, IFN-γ, IL-12, and IL-8 expression levels were significantly increased, which is consistent with the level of expression of NF-κB.2. Clinical results showed that ESR, CRP, and the relative value of neutrophils in-creased, but absolute and relative value of lymphocytes and hemoglobin values decreased between spinal tuberculosis patients and normal control group, and there was significant difference. The microarray results showed there were 68 genes with significant difference between spinal tuberculosis patients and normal control group,16 genes up-regulated, and 52 genes down-regulated. Among the gene, 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. The expressions of TLR-2, p-p38 MAPK, NF-κB, KLF4 were significant increase in the level of mRNA and protein, and there was significant difference.3. However, the expressions of p38 MAPK were no significant increase in the lev-el of protein. TNF-α, IFN-γ, and IL-1 expression levels were also significantly incre-ased after macrophages were treated by 100 ng/ml LPS.Conclusion1. The clinical efficacy of UCCT for spinal tuberculosis was consistent with perip-heral blood gene expressions before and after UCCT, and protein expression of inflammatory cytokines, and NF-κB levels.2. mRNA expression in peripheral blood detected by gene microarray technology can be used to assess the efficacy of UCCT, while the spinal tuberculosis patients were treated by complete debridement and cured by UCCT.3. KLF4 genes participated in anti-tubercular reactions involved in spinal tubercu-losis by regulating monocyte to macrophage differentiation. KLF4 gene may causeinflammation by participating in TLR-2/p38MAPK/NF-κB signaling pathway. |
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