过表达CKLF1基因对强直性脊柱炎滑膜成纤维细胞增殖及成骨转化影响的实验研究
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摘要
目的探索趋化素样因子1 (chemokine-like factor1,CKLF1)对强直性脊柱炎(ankylosingspondyliti,AS)髋关节滑膜成纤维细胞增殖和成骨转化的影响。方法对照组和AS髋关节滑膜组织分别取自我科4例股骨颈骨折和3例重度AS拟行髋关节置换患者。滑膜组织经常规消化后获得单个细胞,并在倒置相差显微镜下进行观察和抗vimentin免疫荧光染色(immunofluorescence,IFC)检测。对上述髋关节滑膜组织及成纤维细胞分别行原位和体外培养,重组腺相关病毒(rAAV)-lacZ、rAAV-hCKLF1转染后21天收集标本。分别采用ELISA、IFC和荧光定量RT-PCR检测细胞增殖、致炎性细胞因子分泌、成骨分化的关键靶基因CKLF1及CCR4的表达。结果第3代正常和AS细胞抗vimentin IFC检测均为阳性,表明分离培养的细胞为成纤维细胞。rAAV-hCKLF1转染后21天,HE染色并计数单位面积下的细胞数、水溶性四氮唑法(WST-1)检测细胞增殖能力和Hoechst 33258检测细胞DNA含量均显示,CKLF1转染促进细胞增殖,且与无病毒转染组、lacZ组相比,差异有统计学意义(P=0.0000,P=0.0260,P=0.0020);正常和AS髋关节滑膜组织及成纤维细胞分泌的致炎性细胞因子(IL-6和TNF-α),均明显升高,且与无病毒转染组、lacZ组相比,差异有统计学意义(P=0.0060、P=0.0020);CKLF1尚能促进AS成纤维细胞表达特异性骨细胞外基质蛋白(OCN和OPN)表达;荧光定量RT-PCR与上述检测结果一致。结论过表达CKLF1促进AS髋关节滑膜成纤维细胞增殖和致炎性细胞因子分泌,增强成骨转化相关靶基因的转录,CKLF1可能在AS关节病理性骨化过程中发挥重要作用。
Objective To investigate the effects of chemokine like factor 1 (CKLF1) gene upon the proliferative activities and osteogenic potentials of hip synovium of ankylosing spondylitis (AS) in situ and in vitro.Methods Normal and AS hip synovium specimens were collected from 4 patients with femoral neck fracture and3 AS patients with severe hip deformities. Synovium specimens were exposed to type II collagenase and obtained single cell suspension, while phase contrast microscopy and anti-vimentin immunofluorescence staining (IFC) were applied to observe the cells. The specimens and fibroblasts were divided and cultured in situ and in vitro respectively, and the recombinant adeno-associated virus (rAAV)-lacZ (E. coli beta-galactosidase gene) and rAAV-hCKLF1 (human CKLF1 cDNA cloned in rAAV-lacZ in place of lacZ) were transduced these samples for 21 days. Cell proliferation (cellularity), secretion of pro-inflammatory cytokines, expression of CKLF1 and CCR4 genes were detected by the water-soluble tetrazolium (WST-1) assay and Hoechst 33258 test (DNA content), enzyme-linked immunosorbent assay (ELISA) assay, IFC test and fluorescent quantitative reverse transcription polymerase chain reaction (RT-PCR),respectively. Results The third passage of normal and AS synovial cells were positive to anti-vimentin, indicating that the cells were synovial fibroblasts. After transduction with rAAV-hCKLF1 for 21 days, cellularity, WST-1 and Hoechst 33258 assays illustrated that CKLF1 gene transfer promoted cell proliferation, compared with the non-viral transduction and lacZ groups (P = 0.0000, P = 0.0260, P = 0.0020). Overexpression of CKLF1 gene enhanced the secretion of pro-inflammatory cytokines (interleukin-6 and tumor necrosis factor-alpha), compared with the nonviral transduction and lacZ groups (P = 0.0060, P = 0.0020) and the expression of bone-specific extracellular matrix proteins. Similar results were observed in fluorescent quantitative RT-PCR test. Conclusions Overexpression of CKLF1 promotes the proliferation of fibroblasts, the secretion of pro-inflammatory cytokines and the expression of osteogenic related target genes, suggesting that CKLF1 might be involved in the pathological ossification of AS.
Objective To investigate the effects of chemokine like factor 1 (CKLF1) gene upon the proliferative activities and osteogenic potentials of hip synovium of ankylosing spondylitis (AS) in situ and in vitro.Methods Normal and AS hip synovium specimens were collected from 4 patients with femoral neck fracture and3 AS patients with severe hip deformities. Synovium specimens were exposed to type II collagenase and obtained single cell suspension, while phase contrast microscopy and anti-vimentin immunofluorescence staining (IFC) were applied to observe the cells. The specimens and fibroblasts were divided and cultured in situ and in vitro respectively, and the recombinant adeno-associated virus (rAAV)-lacZ (E. coli beta-galactosidase gene) and rAAV-hCKLF1 (human CKLF1 cDNA cloned in rAAV-lacZ in place of lacZ) were transduced these samples for 21 days. Cell proliferation (cellularity), secretion of pro-inflammatory cytokines, expression of CKLF1 and CCR4 genes were detected by the water-soluble tetrazolium (WST-1) assay and Hoechst 33258 test (DNA content), enzyme-linked immunosorbent assay (ELISA) assay, IFC test and fluorescent quantitative reverse transcription polymerase chain reaction (RT-PCR),respectively. Results The third passage of normal and AS synovial cells were positive to anti-vimentin, indicating that the cells were synovial fibroblasts. After transduction with rAAV-hCKLF1 for 21 days, cellularity, WST-1 and Hoechst 33258 assays illustrated that CKLF1 gene transfer promoted cell proliferation, compared with the non-viral transduction and lacZ groups (P = 0.0000, P = 0.0260, P = 0.0020). Overexpression of CKLF1 gene enhanced the secretion of pro-inflammatory cytokines (interleukin-6 and tumor necrosis factor-alpha), compared with the nonviral transduction and lacZ groups (P = 0.0060, P = 0.0020) and the expression of bone-specific extracellular matrix proteins. Similar results were observed in fluorescent quantitative RT-PCR test. Conclusions Overexpression of CKLF1 promotes the proliferation of fibroblasts, the secretion of pro-inflammatory cytokines and the expression of osteogenic related target genes, suggesting that CKLF1 might be involved in the pathological ossification of AS.
引文
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[18]曾晖,肖德明,陶可,等.TGF-β1对骨髓干细胞分化过程中Wnt信号通路相关基因表达的影响[J].中华实验外科杂志,2013,30(7):1347-1350.
[19]Tan YX,Han WL,Chen YY,et al.Chemokine-like factor 1,a novel cytokine,contributes to airway damage,remodeling and pulmonary fibrosis[J].Chin Med J(Engl),2004,117(8):1123-1129.
[20]Bal A,Unlu E,Bahar G,et al.Comparison of serum IL-1beta,sIL-2R,IL-6,and TNF-alpha levels with disease activity parameters in ankylosing spondylitis[J].Clin Rheumatol,2007,26(2):211-215.
[21]Gratacós J,Collado A,Filella X,et al.Serum cytokines(IL-6,TNF-alpha,IL-1 beta and IFN-gamma)in ankylosing spondylitis:a close correlation between serum IL-6 and disease activity and severity[J].Br J Rheumatol,1994,33(10):927-931.
[22]Ke X,Jia L,Jing H,et al.Effects of novel human chemokinelike factor 1(CKLF1)on bone marrow hematopoietic stem cell/progenitor cell in vitro[J].Zhonghua Xue Ye Xue Za Zhi,2002,23(6):301-303.
[23]Xuan CR,Chen Y,He PY,et al.Chemoattractive effects of chemokine-like factor 1 on human arterial smooth muscle cells[J].Beijing Da Xue Xue Bao,2009,41(2):148-151.
[24]Dalby MJ,Gadegaard N,Tare R,et al.The control of human mesenchymal cell differentiation using nanoscale symmetry and disorder[J].Nat Mater,2007,6(12):997-1003.
[2]Qin Y,He LD,Sheng ZJ,et al.Increased CCL19 and CCL21levels promote fibroblast ossification in ankylosing spondylitis hip ligament tissue[J].BMC Musculoskelet Disord,2014,15:316.
[3]Zou YC,Yang XW,Yuan SG,et al.Downregulation of dickkopf-1 enhances the proliferation and osteogenic potential of fibroblasts isolated from ankylosing spondylitis patients via the Wnt/β-catenin signaling pathway in vitro[J].Connect Tissue Res,2016,57(3):200-211.
[4]Grisar J,Bernecker PM,Aringer M,et al.Ankylosing spondylitis,psoriatic arthritis,and reactive arthritis show increased bone resorption,but differ with regard to bone formation[J].J Rheumatol,2002,29(7):1430-1436.
[5]Yu F,Cui Y,Zhou X,et al.Osteogenic differentiation of human ligament fibroblasts induced by conditioned medium of osteoclast-like cells[J].Biosci Trends,2011,5(2):46-51.
[6]Braun J,Bollow M,Neure L,et al.Use of immunohistologic and in situ hybridization techniques in the examination of sacroiliac joint biopsy specimens from patients with ankylosing spondylitis[J].Arthritis Rheum,1995,38(4):499-505.
[7]Han W,Lou Y,Tang J,et al.Molecular cloning and characterization of chemokine-like factor 1(CKLF1),a novel human cytokine with unique structure and potential chemotactic activity[J].Biochem J,2001,357(Pt 1):127-135.
[8]李虎,吕厚山,陈占昆,等.趋化素样因子1在类风湿关节炎滑膜中的表达研究[J].中华风湿病学杂志,2007,11(2):74-77.
[9]Tao K,Tang X,Wang B,et al.Distinct expression of chemokine-like factor 1 in synovium of osteoarthritis,rheumatoid arthritis and ankylosing spondylitis[J].J Huazhong Univ Sci Technolog Med Sci,2016,36(1):70-76.
[10]李虎,李儒军,曹宸喜,等.趋化素样因子1过表达促进强直性脊柱炎髋关节韧带成纤维细胞增殖和向成骨细胞转化的实验研究[J].中华风湿病学杂志,2017,21(9):614-621.
[11]Dougados M,van der Linden S,Juhlin R,et al.The Euro-pean Spondylarthropathy Study Group preliminary criteria for the classification of spondylarthropathy[J].Arthritis Rheum,1991,34(10):1218-1227.
[12]Tao K,Zeng H,Xiao DM,et al.Influences of IL-6R antibody on PMMA bone cement-mediated expression of OPG and RANKL in synovial fibroblasts[J].J Huazhong Univ Sci Technolog Med Sci,2014,34(2):241-246.
[13]Riera KM,Rothfusz NE,Wilusz RE,et al.Interleukin-1,tumor necrosis factor-alpha,and transforming growth factorbeta 1 and integrative meniscal repair:influences on meniscal cell proliferation and migration[J].Arthritis Res Ther,2011,13(6):R187.
[14]Samulski RJ,Chang LS,Shenk T.A recombinant plasmid from which an infectious adeno-associated virus genome can be excised in vitro and its use to study viral replication[J].J Virol,1987,61(10):3096-3101.
[15]Samulski RJ,Chang LS,Shenk T.Helper-free stocks of recombinant adeno-associated viruses:normal integration does not require viral gene expression[J].J Virol,1989,63(9):3822-3828.
[16]Cucchiarini M,Thurn T,Weimer A,et al.Restoration of the extracellular matrix in human osteoarthritic articular cartilage by overexpression of the transcription factor SOX9[J].Arthritis Rheum,2007,56(1):158-167.
[17]McClure C,Cole KL,Wulff P,et al.Production and titering of recombinant adeno-associated viral vectors[J].J Vis Exp,2011,(57):e3348.
[18]曾晖,肖德明,陶可,等.TGF-β1对骨髓干细胞分化过程中Wnt信号通路相关基因表达的影响[J].中华实验外科杂志,2013,30(7):1347-1350.
[19]Tan YX,Han WL,Chen YY,et al.Chemokine-like factor 1,a novel cytokine,contributes to airway damage,remodeling and pulmonary fibrosis[J].Chin Med J(Engl),2004,117(8):1123-1129.
[20]Bal A,Unlu E,Bahar G,et al.Comparison of serum IL-1beta,sIL-2R,IL-6,and TNF-alpha levels with disease activity parameters in ankylosing spondylitis[J].Clin Rheumatol,2007,26(2):211-215.
[21]Gratacós J,Collado A,Filella X,et al.Serum cytokines(IL-6,TNF-alpha,IL-1 beta and IFN-gamma)in ankylosing spondylitis:a close correlation between serum IL-6 and disease activity and severity[J].Br J Rheumatol,1994,33(10):927-931.
[22]Ke X,Jia L,Jing H,et al.Effects of novel human chemokinelike factor 1(CKLF1)on bone marrow hematopoietic stem cell/progenitor cell in vitro[J].Zhonghua Xue Ye Xue Za Zhi,2002,23(6):301-303.
[23]Xuan CR,Chen Y,He PY,et al.Chemoattractive effects of chemokine-like factor 1 on human arterial smooth muscle cells[J].Beijing Da Xue Xue Bao,2009,41(2):148-151.
[24]Dalby MJ,Gadegaard N,Tare R,et al.The control of human mesenchymal cell differentiation using nanoscale symmetry and disorder[J].Nat Mater,2007,6(12):997-1003.