| CHAPTER â… Th17cell in blood and bone tissue of human spinal tuberculosis disease and its phenotypeObjective To investigate the spinal tuberculosis in patients with disease of bone tissue, peripheral blood is present in the Th17cell, its related cytokines changes, and to explore its number, phenotype and transcription factors retinoic acid related orphan receptor T (ROR y t expression).Methods10cases of spinal tuberculosis debridement, interbody decompression and internal fixation combined with antituberculotic treatment of patients at admission, intraoperative and postoperative1weeks blood specimens, spinal tuberculosis bone and extraction of autologous iliac bone graft block specimens. Histological observation of tuberculosis of bone and extraction of autogenous bone grat tissue morphological changes. Flow cytometry and double immunofluorescence staining identified specimens of Th17cells in the presence of its share in CD4+T cell percentage, and determine the phenotype. Enzyme linked immunosorbent assay for the detection of samples in the interferon (IFN-y) and tumor necrosis factor (TNF alpha) level; PCR method for the detection of samples in the ROR gamma tmRNA expression.Results (1) blood specimens before and after the changes at the time of admission IFN gamma168.7+19.2pg/L intraoperative IFN gamma113.7+15.3pg/L, after1weeks of IFN gamma62.7+14.9pg/L; admission TNF alpha37.1+8.4ng/mL, TNF in art-alpha32.1+5.3ng/mL, TNF postoperative alpha0.2+7.4ng/mL. Preoperative and intraoperative comparison of increased significantly (P<0.05), intraoperative and postoperative comparison were significantly higher (P<0.05);(2) spinal tuberculosis bone and extraction of autologous iliac bone graft block specimen contrast changes disease in bone tissue of IFN gamma78.7+32.4pg/L, autogenous iliac bone grafting bone block IFN gamma11.3+5.2pg/L; disease of bone tissue in TNF-a9.6+4.4ng/mL, autologous iliac bone graft block TNF alpha7.5+4.3ng/mL, spinal tuberculosis bone was significantly higher than that of autologous bone grafting block concentration (P<0.05);(3) in bone tissue and blood IFN-y and TNF-a level comparison of spinal tuberculosis bone tissue than on admission and intraoperative, postoperative blood concentrations increased (P<0.05), extraction of autologous iliac bone graft block concentrations were preoperative and intraoperative blood concentration reduce (P<0.05), andl weeks after operation, no significant difference (P>0.05);(4) the proportion of Th17cells, ROR y t and IL-17mRNA expression changes of preoperative blood Thl7cell for CD4+T lymphocyte ratio was0.8%+0.1%,0.9%+0.2%in operation,1week after operation was0.4%+0.1%, preoperative and intraoperative comparison of increased significantly (P<0.05), intraoperative and postoperative comparison were significantly higher (P<0.05). Spinal tuberculosis bone; Th17cell ratio:8.2%+3.2%, autologous iliac bone graft block Th17cell ratio:0.2%-0.1%. Spinal tuberculosis bone was significantly higher than that of autologous bone grafting block concentration (P <0.05). Preoperative blood ROR y t expression amount of (23.4+3.6) ng/L tuberculosis in bone tissue of ROR gamma t expression amount of (56.3+4.4) ng/L, autologous iliac bone graft block ROR gamma t expression amount of (2.1+0.2) ng/L, tuberculosis bone tissue concentration higher blood and autologous iliac bone graft block concentration (P<0.05);(5) Thl7cell phenotype of spinal tuberculosis bone in Th17cells expressing CD45RO phenotypic characteristics ratio of87.4%+6.8%,5.9%+1.8%CD45RA expression, compared with statistical difference (P<0.05); blood Thl7cells expressing CD45RO phenotypic characteristics ratio of69.3%+5.4%,4.3%+2%CD45RA expression, compared with statistical difference (P<0.05).Conclusion spinal tuberculosis bone and blood in the presence of Th17cells, and in spinal tuberculosis bone in concentration, its phenotypic characteristics for memory T lymphocyte. CHAPTER IITh17cells in the blood and disease of spinal tuberculosis bone tissue is upregulated in related researchObjective To investigate the role of Th17cells by cytokines and chemokines types, discusses Thl7source.Methods10cases of spinal tuberculosis debridement, interbody decompression and internal fixation combined with antituberculotic treatment of patients at admission, intraoperative and postoperative1weeks blood specimens, spinal tuberculosis bone and extraction of autologous iliac bone graft block specimens. Enzyme linked immunosorbent assay (ELISA) detection in specimens from IFN-y, IL-1beta, IL-6, IL-23and TGF-a concentration; preparation of purified primitive CD4+T lymphocytes and IL-1, IL-6, IFN beta gamma, TNF beta alone or mixed culture after the counting Th17cell percentage change; by ELISA assay osteoblastic expression of CCL20, CCL22, CCL27concentration, immunofluorescence staining and confocal laser scanning microscopy observation of osteogenic cells expressed CCL20, CCL22, CCL27morphology; observation of Thl7chemotactic index change. Flow cytometric study of tuberculosis of bone and blood Thl7cells expressing the CCR receptor and CCL ligand relationship.Results (1), spinal tuberculosis before admission blood group IL-1beta1437+73pg/L, during operation for1167+81.6pg/L,1weeks after operation blood IL-lbeta761.3+57.8pg/L; spinal tuberculosis before admission blood group IL-6was784.1+ 87.7pg/L,intraoperative was693.5+85.7pg/L,1week afer operation was217.8+97.5pg/Lï¼› spinal tuberculosis group before admission blood IL-23258.4+64.7pg/L,intraoperative was254.2+54.1pg/L,1week after operation was126.6+92.4pg/Lï¼›spinal tuberculosis before admission blood group CCL20was31.3+2.5pg/L,intraoperative was54.2+7.1pg/L,after lweek of26.5+9.9pg/Lï¼›spinal tuberculosis bone in CCL20831.3+2.5pg/L, CCL22was947.1+7.1pg/L,CCL27was932.8+5.2pg/Lï¼›autogenous iliac bone block CCL20was16.1+2.4pg/L,CCL22was12.5+0.4pg/L,CCL27132.8+5.2pg/Lï¼›IL-6,IL-23and TGF beta concentration were significantly higher(P<0.01).Tuberculosis group in Th17cell ratio and IFN-y,IL-1,IL-6, IL-23and TGF beta beta concentration between both have significantly positive correlation(P<0.05).Tuberculosis of bone in the ligand chemokine CCL20, CCL22and CCL27were significantly higher than that of the blood concentration and extraction of the iliac bone autograft in the concentration(P<0.05).(2),spinal tuberculosis bone Th17cell surface expression of CCR4cell ratio:67.3ï¼…+2.3ï¼…,CCR6:72.4%+4.1ï¼…,CCR10:56.8%+2.3ï¼…,CCR2:2.4%+0.3ï¼…,CCR3:21.5ï¼…+4.6ï¼…,3.7%+0.5ï¼…CCR5.Preoperative blood Th17cell surface expression of CCR4cell ratio:54.3ï¼…+6.2ï¼…,CCR6:69.3ï¼…+6.2%, CCR10:52.5%+7.1ï¼…,CCR2:3.8%+1.5%,CCR3:24.1ï¼…+6.8%,2.2%+1.4%CCR5.Tuberculosis of bone in Th17cell chemokine receptor expression was significantly higher than that of the blood(P<0.05).(3),Th17chemotactic index changes alone to join alpha CCL20Th17cells and osteoblasts chemotaxis are weakened, joined alpha CCL20, alpha CCL22and alpha CCL27Th17ceEll chemotaxis index decreased significantly.Conclusion Tuberculosis of bone tissue and blood of chemokine receptor and its ligand on inflammatory factor upregulation, which IL-6, IL-1, TNF alpha beta upregulation of Th17cell differentiation, and IFN gamma inhibition of Th17up-regulation of Spinal tuberculosis bone in Th17cells derived from two sources:1, their own local tissue differentiation of Thl7cells proliferationï¼›2, in the CCL20-CCR6chemotactic axes, CCL22-CCR4and CCL27-CCR10chemokine axis function and osteoblast their chemotaxis, Thl7cells from the blood raise to disease in bone tissue. CHAPTER IIspinal tuberculosis bone osteoblast in antigen presentation and Immune regulation function of related researchObjective To investigate the spinal tuberculosis bone in Thl7cells to osteoblast proliferation and osteogenic cells on Th17cell antigen of.Methods10cases of spinal tuberculosis patients in experimental group, using a modified enzyme digestion method to obtain the spinal tuberculosis bone in osteogenesis cells after culture, to be osteoblasts in culture dish fusion tiled to pipette in the central draw distance gap into bone cell injury model, IL-17, IL-22, IFN-ray and its the corresponding antibody respectively and damage model of culture, were observed at different time points into bone cell fusion. Osteoblasts to alkaline phosphatase test and alizarin red testing; flow cytometer in spinal tuberculosis bone osteoblastic cells and extraction of the iliac bone autograft bone cell surface HLA-DR, CD80and CD86, in blood purification of CD4+T lymphocytes with or without tuberculosis antigen85B in cases with IL-17, IL-22, osteogenesis cell, IFN-y and resistance to CD80, CD86monoclonal antibody combined mixed culture, detection of T cell proliferation marker Ki-67+CD4+T cell number and Th17number.ResuIt (1), in a tuberculosis bone in isolated osteoblasts express the IL-22R ratio of59.6%+4.6%,68.9%+7.2%IL-17R, IFN y R was51.9%+5.9%;(2), IL-22, IL-17promotes osteoblastic cell proliferation and viability, and IFN gamma inhibition of osteoblast and vitality. Anti-IL-22, anti-IL-17inhibits the proliferation of osteoblasts, and anti-IFN-y promote osteoblast proliferation;(3), separated from tuberculosis bone osteoblastic cells expressing HLA-DR ratio was90.4%+3.9%,87.3%+4.1%CD80expression, expression of CD86was93.2%+7.2%, in Mycobacterium tuberculosis specific antigen85B under the conditions of existence, Ki-67+CD4+T cells decreased significantly, tuberculosis bone single cell suspensions of Ki-67+CD4+T cells (with osteoblasts condition) significantly increased;(4), Thl7cells in the osteoblastic cells and the presence of85B increased, IL-17and IL-22, IFN-y can significantly increase the number of Th17cells, CD80resistance anti CD86and CTLA-41g, so that Thl7cells downregulate.Conclusion Th17cells promote osteogenic cell injury and proliferation; spinal tuberculosis bone in osteogenesis cells with antigen-presenting ability, and by antigen presenting information to promote the proliferation of CD4+T lymphocytes; Osteoblasts antigen results in the stimulation of Th17cell development and differentiation. |
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