IL-6R抗体调节PMMA骨水泥介导的滑膜成纤维细胞MMPs和血管化因子mRNA表达

Regulation of IL-6R antibody on PMMA bone cement mediated mRNA expression of MMPs and vascular factors by synovial fibroblasts

目的探讨IL-6R抗体对PMMA骨水泥介导的滑膜成纤维细胞MMP-1和MMP-3、VEGF和PDGF mRNA表达的调节。方法从全髋关节置换术中获取滑膜组织消化并传代培养。倒置相差显微镜对滑膜细胞进行形态学观察,免疫细胞化学(SABC法)染色对滑膜成纤维细胞进行鉴定。根据加入不同培养物,实验分为3组:PMMA组:75μg/mL PMMA骨水泥颗粒;IL-6R抗体组:10 ng/mL IL-6R抗体+75μg/mL PMMA骨水泥颗粒;空白对照组。采用细胞计数试剂盒-8(Cell Counting Kit-8,CCK-8)检测IL-6R抗体、PMMA对滑膜成纤维细胞增殖活力影响;实时荧光定量PCR(real timefluorescence quantitative polymerase chain reaction,FQ-PCR)检测MMP-1、MMP-3、VEGF和PDGF mRNA表达。结果原代滑膜细胞贴壁后,初期为短梭形。连续3次传代后,95%以上细胞为长梭形成纤维细胞样细胞。SABC法染色检测结果显示:上述成纤维样细胞抗CD68抗体阴性,抗vimentin抗体呈棕黄色,为阳性反应。CCK-8检测显示:与PMMA组和空白对照组相比,IL-6R抗体组吸光度(A)值明显降低(P<0.01);而空白对照组与PMMA组间吸光度(A)值无统计学差异(P>0.05)。FQ-PCR检测发现:与PMMA组和空白对照组相比,IL-6R抗体组中MMP-1和MMP-3、VEGF和PDGF mRNA的表达明显受到抑制(P<0.01);PMMA组中MMP-1和MMP-3、VEGF和PDGF表达较空白对照组升高(P<0.05)。结论 IL-6R抗体能显著抑制滑膜成纤维细胞MMP-1和MMP-3、VEGF和PDGFmRNA的表达,为生物制剂预防和治疗假体无菌性松动提供分子生物学的理论依据。

Objective To investigate the PMMA bone cement-mediated expression of MMP-1, MMP-3, VEGF, and PDGF mRNA in synovial fibroblasts regulated by IL-6R antibody. Methods The synovial tissue obtained from total hip arthroplasty was digested and subcuhured. Phase-contract microscope was employed to observe the synovial cells and immunocytochcmistry （SABC method） staining was used to identify synovial fibroblasts. The experiment was divided into 3 groups according to the different supplement in cultures： PMMA group （75μg/ml PMMA bone cement particles） ; IL-6R antibody group （10 ng/ml IL- 6R antibody and 75 μg/ml PMMA bone cement particles） ; and the control group. Cell proliferation activity regulated by IL-6R antibody or PMMA was measured by using a cell counting kit-8 （CCK-8）. FQ-PCR was used to detect the mRNA expressions of MMP-1, MMP-3, VEGF, and PDGF. Results The primary synovial cells showed a short spindle shape after adhesion. After 3 consecutive passages, more than 95% of the cells became fibroblast-like cells with a long spindle shape. SABC staining showed that the anti-CD68antibody was negative and anti-vimentin antibody was positive in fibroblast-like cells. CCK-8 test demonstrated that the absorbance value （A） was significantly lower in IL-6R antibody group than that in PMMA group and control group （P 〈 0. 01 ）. The A value was not statistically different between the control group and PMMA group （ P 〉 0. 05 ）. FQ-PCR measurement showed that mRNA expressions of MMP-1, MMP-3, VEGF, and PDGF in IL-6R antibody group were significantly inhibited than those in PMMA group and control group （P 〈 0. 01 ）. The mRNA expressions of MMP-1, MMP-3, VEGF, and PDGF in PMMA group were higher than those in control group （P 〈 0.05）. Conclusion IL-6R antibody could significantly inhibit the mRNA expressions of MMP-1, MMP-3, VEGF, and PDGF in synovial fibroblasts. This may provide theoretical basis of molecular biology for the prevention and treatment of aseptic loosening of the prosthesis.