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  

Recombinant adeno-associated virus-mediated chemokine like factor 1 gene transferring modulates the proliferative activities and osteogenic potentials of human hip ligaments of ankylosing spondylitis patients

作　　者：李虎[1] 李儒军[1] 曹宸喜 柯岩[1] 林剑浩[1] 陶可[1] Li Hu Li Rujun Cao Chenxi Ke Yon Lin Jianhao Tao Ke(Institute of Arthritis, Peking University People＇s Hospital, Beijing, 100044, China)

机构地区：[1]北京大学人民医院骨关节科关节炎诊疗中心,100044

出　　处：《中华风湿病学杂志》2017年第9期614-621,I0002,共9页Chinese Journal of Rheumatology

基　　金：国家自然科学基金（81641175,81672183）;北京大学人民医院院内发展基金（RDB2015-10,RDY2016-15）;2015年国际骨关节炎研究协会（OARSI）访问学者奖学金;2016年国际软骨修复胁会（ICRS）奖学金;2016年北京厚爱关节健康公益基金会青年学者奖学金;2017年德国骨科医师大会（DKOU）奖学金

摘　　要：目的探索趋化素样因子1（CKLF1）对AS髋关节韧带成纤维细胞增殖和向成骨细胞转化的影响。方法正常和AS髋关节韧带组织分别取自我科6例股骨颈骨折和4例重度AS拟行髋关节置换的患者。组织经常规消化得单细胞，并行倒置相差显微镜观察和抗波形蛋白（vimentin）免疫荧光染色（IFC）检测。对上述髋关节韧带组织及成纤维细胞分别行原位（in situ）和体外（in vitro）培养，重组腺相关病毒（rAAV）-lacZ、rAAV-hCKLF1转染21 d后收集标本。细胞增殖、致炎性细胞因子分泌、成骨关键靶基因、CKLF1及CCR4表达等分别采用ELISA、IFC和荧光定量反转录（RT）-PCR检测。各组间数据用单因素方差分析（LSD法），2组间比较采用t检验。结果第2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21 d后，苏木精-伊红（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[与无病毒转染组、lacZ组相比，差异具有统计学意义（F=6.98，64.32，115.91；P〈0.05或P〈0.01）]；正常和AS髋关节韧带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[与无病毒转染组、lacZ组相比，差异具有统计学意义（F=34.57，8.89；P〈0.05或P〈0.01）]；此外，与无病毒转染组、lacZ组及正常成纤维细胞的各组相比，CKLF1尚能促进AS成纤维细胞表达特异性骨细胞外基质蛋白[骨桥蛋白（OPN）和骨钙蛋白（OCN）]；荧光定量RT-PCR也得到了与上述检测结果相近的发现。结论过表达CKLF1促进成纤维细胞增殖和致炎性细胞因子分泌，增强成骨相关靶基因的转录，推测CKLF1可能在AS关节病理性骨化过程中发挥重要作用。To investigate the effects of chemokine like factor 1（CKLF1） gene on the proliferative activities and osteogenic potentials of hip ligaments of ankylosing spondylitis （AS） in situ and in vitro.MethodsNormal and AS hip ligament specimens were collected from 6 patients with femoral neck fracture and 4 AS patients with severe hip deformities. Ligament specimens were exposed to type Ⅱ colla-genase and obtained a single cell suspension, while phase contrast microscopy and anti-vimentin immuno-fluorescence 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 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）, IFC test and fluorescent quantitative reverse transcription polymerase chain reaction （RT-PCR）, respectively. Statistical analysis significance was conducted using the Student＇s t test and one-way analysis of variance （ANOVA） （LSD） test where appropriate.ResultsThe second passage of normal and AS cells were positive for anti-vimentin, indicating that the cells were fibroblasts. After transducing 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, F=6.98, 64.32, 115.91, P〈0.05 or P〈0.01）. Overexpression of CKLF1 gene enhanced the secretion of pro-inflammatory cytokines （interleukin-6 and tumor necrosis factor-alpha） and the expression of bone- specific extracellular matrix proteins （osteopontin and osteocalcin） （F=34.57, 8.89, P〈

关 键 词：脊柱炎 强直性 骨化 异位性 成纤维细胞 转染 趋化素样因子1 

分 类 号：R593.23[医药卫生—内科学]