摘要
目的:探讨昼夜节律关键基因BMAL1在小鼠骨折愈合过程中的作用。方法:构建40只BMAL1基因敲除(KO)与野生型(WT)小鼠股骨骨折模型,采用髓内钉固定,并在术后第7、14、21、28天取材。通过Micro CT三维重建,定量分析骨体积分数(BV/TV)、骨小梁厚度(Tb.Th)、骨小梁数目(Tb.N)、骨小梁间距(Tb.Sp)以及组织矿物密度(TMD),观察骨痂形成及骨结构变化。结合苏木精-伊红染色法与甲苯胺蓝染色,评估组织学水平的骨修复及软骨转化情况;并通过免疫荧光染色检测关键成骨与成软骨相关蛋白[骨桥蛋白(BSP)、Ⅱ型胶原(COL-Ⅱ)及抗聚集蛋白多糖(ACAN)]的表达差异。结果:BMAL1缺失显著延缓新骨形成。Micro-CT结果显示,KO组骨痂形成薄弱,在骨折愈合的第21天较WT组显示出软骨分化异常或不完全成熟;KO组的BV/TV、Tb.Th、Tb.N均低于WT组,Tb.Sp显著升高(P<0.05)。组织学结果表明,WT组在术后第14天已出现结构清晰的骨小梁及成熟软骨;而KO组仍以纤维组织为主,伴随炎症延长及软骨形成不足。第28天,WT组的骨痂基本完成骨化,KO组仍存在大量未骨化软骨。免疫荧光结果显示,BMAL1的缺失显著降低BSP、COL-Ⅱ及ACAN的表达水平。结论:BMAL1缺失会显著延缓骨折愈合,表现为骨痂形成不足、软骨转化受阻及成骨与成软骨相关蛋白表达下降。
Objective To explore the role of the circadian rhythm key gene BMAL1 in the fracture healing process in mice.Methods Femoral fracture models of 40 BMAL1 figene knockout(KO)and wild-type(WT)mice were constructed and fixed with intramedullary nails,and then samples were collected on the 7th,14th,21st,and 28th days after the operation.Micro-CT three-dimensional reconstruction was used to quantitatively analyze changes of bone volume/total volume(BV/TV),trabecular thickness(Tb.Th),trabecular number(Tb.N),trabecular separation(Tb.Sp),and tissue mineral density(TMD),and the formation of callus and changes of bone structure were observed.Bone repair and cartilage transformation at the histological level were evaluated combination of hematoxylin and eosin staining and toluidine blue staining,and immunofluorescence staining was used to detect the expression differences of key osteogenic and chondrogenic related proteins[bone sialoprotein(BSP),collagen typeⅡ(COL-Ⅱ),and anti-aggrecan(ACAN)].Results BMAL1 deficiency significantly delayed new bone formation.Micro-CT results showed that callus formation in the KO group was weak,and the cartilage differentiation was abnormal or incompletely mature on the 21st day of fracture healing compared with that in the WT group.Compared with those in the WT group,all bone structure parameters decreased significantly compared with the WT groupvalues of BV/TV,Tb.Th,Tb.N were lower,while the Tb.Sp value was significantly increased in the KO group(P<0.05).The histological results indicated that clearly structured trabeculae and mature cartilage had appeared in the WT group on the 14th day after the operation,while the KO group was still dominated by fibrous tissue,accompanied by prolonged inflammation and insufficient cartilage formation.On the 28th day,the callus in the WT group had basically completed ossification,while there was still a large amount of unossified cartilage in the KO group.The immunofluorescence results showed that the BMAL1 fifideficiency significantly reduced the expressions of BSP,COL-Ⅱand ACAN.Conclusions BMAL1 deficiency may significantly delay fracture healing,which is manifested by insufficient callus formation,hindered cartilage transformation,and decreased expressions of osteogenic/chondrogenic related proteins.
作者
钟研
齐凤
郑凝浩
王鸿博
薛徽
ZHONG Yan;QI Feng;ZHENG Ninghao;WANG Hongbo;XUE Hui(Cental Laboratory:the Fist Afiliated Hospital of Qiqihar Medical University Qiqihar,Heilongjiang 161006,China)
出处
《影像研究与医学应用》
2025年第24期28-33,共6页
Journal of Imaging Research and Medical Applications
基金
黑龙江省自然科学基金项目(PL2024H254)
齐齐哈尔市科技计划联合引导项目(LSFGG-2023051)。
