摘要
目的探讨在根尖囊肿中含蛋白结构域A的纤维连接蛋白(EDA+FN)与成纤维细胞破骨作用之间的关系。方法采用免疫组织化学法对根尖囊肿标本进行染色,分析EDA+FN染色强度与根尖囊肿颌骨病变范围的关系。分别培养从根尖囊肿标本(研究组,20例)和正常颌骨标本(对照组,6例)分离出的成纤维细胞,并制作条件培养基,诱导破骨细胞形成。用CRISPR/Cas9系统敲除成纤维细胞的蛋白结构域A(EDA)外显子,比较EDA敲除组与非EDA敲除组破骨相关因子mRNA表达水平。结果EDA+FN的染色强度与根尖囊肿颌骨病变范围呈正相关(r=0.594,P=0.006)。研究组EDA+FN mRNA的表达水平明显高于对照组(P<0.05)。研究组成纤维细胞诱导的破骨细胞数与EDA+FN mRNA表达水平呈正相关(r=0.817,P<0.001)。EDA敲除组成纤维细胞诱导的破骨细胞数明显少于非EDA敲除组(P<0.05)。EDA敲除组成纤维细胞中白细胞介素-6、巨噬细胞集落刺激因子、肿瘤坏死因子-α、血管内皮生长因子A、核因子-κB受体活化因子配体(RANKL)和RANKL/骨骼保护素的mRNA表达水平均低于非EDA敲除组(P<0.05)。结论根尖囊肿成纤维细胞能分泌EDA+FN,促进骨破坏,而敲除EDA外显子可抑制骨破坏。
Objective To explore the relationship between fibronectin containing protein domain A(EDA+FN)and osteoclasty of fibroblast in radicular cyst.Methods Immunohistochemistry was used to stain the radicular cyst specimens,and the relationship between the intensity of EDA+FN staining and the range of jaw lesions in radicular cyst was analyzed.Cultured fibroblasts isolated from radicular cyst specimens(study group,20 cases)and normal jaw specimens(control group,6 cases),and conditioned medium was prepared to induce osteoclast generation.The EDA exons of fibroblasts were knocked out by the CRISPR/Cas9 system,and the mRNA expression levels of osteoclast-related factors were compared between EDA knockout group and non-EDA knockout group.Results There was a positive correlation between the staining intensity of EDA+FN and the range of jaw lesions in radicular cyst(r=0.594,P=0.006).The expression level of EDA+FN mRNA in the study group was significantly higher than that in the control group(P<0.05).The number of osteoclasts induced by fibroblasts in the study group was positively correlated with the expression level of EDA+FN mRNA(r=0.817,P<0.001).The number of osteoclasts induced by fibroblasts in the EDA knockout group was significantly lower than that in the non-EDA knockout group(P<0.05).The mRNA expression levels of interleukin-6,macrophage colony stimulating factor,tumor necrosis factor-α,vascular endothelial growth factor A,nuclear factorκB receptor activator ligand(RANKL)and RANKL/osteoprotegerin in fibroblasts of EDA knockout group were lower than those in non-EDA knockout group(P<0.05).Conclusion The fibroblasts of radicular cyst can secrete EDA+FN and promote bone destruction,however,EDA exons knockout can inhibit bone destruction.
作者
陈柚杉
王海丞
CHEN Youshan;WANG Haicheng(Department of Clinical Laboratory,Tongji University Affiliated Stomatological Hospital,Shanghai 200072,China;College of Stomatology Affiliated to Tongji University/Shanghai Engineering Research Center of Tooth Restoration and Regeneration,Shanghai 200072,Chin)
出处
《检验医学与临床》
CAS
2020年第24期3553-3556,共4页
Laboratory Medicine and Clinic
基金
国家自然科学基金青年科学基金项目(81600836)。
