沉默Hoxa9基因可促进胫骨骨折后的成骨分化及骨折愈合  

Hoxa9 silencing promotes tibial fracture healing by regulating osteogenic differentiation

作　　者：张超[1] 李兴勇 马贵福[1] 普星宇 骆文远[1] Zhang Chao;Li Xingyong;Ma Guifu;Pu Xingyu;Luo Wenyuan(Department of Orthopedics,Gansu Provincial Hospital,Lanzhou 730000,Gansu Province,China)

机构地区：[1]甘肃省人民医院骨三科,甘肃省兰州市730000

出　　处：《中国组织工程研究》2020年第35期5600-5606,共7页Chinese Journal of Tissue Engineering Research

摘　　要：背景:研究表明,Hoxa10基因受到miRNA的调节后与成骨分化存在一定的关联性,但有关Hoxa9在成骨分化和胫骨骨折愈合中的表达和功能尚未见报道。目的:初步探讨Hoxa9对体外骨髓间充质干细胞成骨分化的影响及其体内模型的骨折损伤的治疗作用。方法:①体外实验:在成骨诱导培养基中培养小鼠骨髓间充质干细胞,分别进行Hoxa9基因被shHoxa9沉默或被MSCV-Hoxa9过表达处理;②体内实验:构建胫骨骨折模型大鼠,分别用Hoxa9和shHoxa9干预。结果与结论:①体外实验qRT-PCR及Western blot检测显示,Hoxa9的过表达能够明显抑制骨髓间充质干细胞的成骨分化(P<0.05),细胞中成骨相关基因标记骨钙素、骨桥蛋白、Runx2和胶原蛋白Ⅰ的mRNA及蛋白均下调(P<0.05),细胞中钙化结节的数量、成骨分化能力及碱性磷酸酶活性显著降低(P<0.05),而沉默Hoxa9的表达可逆转上述现象;②体内实验组织学染色结果表明,经Hoxa9干预的大鼠组织中骨性损伤明显增加,未发现组织间胶原纤维和愈合损伤组织内的软骨,shHoxa9干预大鼠显示骨的大小和数量明显增加,检测到组织间胶原纤维和愈合损伤组织内的软骨;③体内实验qRT-PCR及Western blot检测发现,Hoxa9干预的大鼠骨折损伤组织中成骨相关基因标记骨钙素、骨桥蛋白、Runx2和胶原蛋白Ⅰ的mRNA及蛋白均下调(P<0.05);而shHoxa9组大鼠骨折损伤组织中成骨相关基因标记骨钙素、骨桥蛋白、Runx2和胶原蛋白Ⅰ的mRNA及蛋白均上调(P<0.05);④上述数据说明,沉默Hoxa9基因可促进骨髓间充质干细胞的成骨分化,进而促进骨折愈合。BACKGROUND:Studies have shown that Hoxa10 gene has a certain correlation with osteogenic differentiation after microRNA regulation.However,the expression and function of Hoxa9 in osteogenic differentiation and tibial fracture healing have not been reported.OBJECTIVE:To preliminarily investigate the effects of Hoxa9 on osteogenic differentiation of bone marrow mesenchymal stem cells in vitro and on fracture healing in an in vivo model.METHODS:In vitro experiment:In bone marrow mesenchymal stem cells cultured in osteogenic induction medium,Hoxa9 was inhibited by shHoxa9 or overexpressed by MSCV-Hoxa9.In vivo experiment:A tibial fracture model was constructed in rats,followed by intervention with Hoxa9 and shHoxa9.RESULTS AND CONCLUSION:In the in vitro test,qRT-PCR and western blot results indicated that Hoxa9 overexpression could significantly inhibit the osteogenic differentiation of bone marrow mesenchymal stem cells(P<0.05),downregulate osteogenesis-related gene markers osteocalcin,osteopontin,Runx2 and collagen type I at mRNA and protein levels(P<0.05),and reduce the number of calcified nodules,osteogenic differentiation ability and alkaline phosphatase activity(P<0.05).In contrast,transfection of BMSCs with silenced Hoxa9 gene could reverse the above indicators.In the in vivo test,histological staining results showed that Hoxa9 intervention significantly aggravated bone damage in rats,and no interstitial collagen fibers and cartilage in the callus were found.The rats intervened by shHoxa9 showed a significant increase in bone size and number,and inter-tissue collagen fibers and cartilage in callus were detected.Findings from qRT-PCR and western blot in the in vivo test showed that the mRNA and protein expression of osteocalcin,osteopontin,Runx2 and collagen type I were significantly down-regulated in the Hoxa9 group(P<0.05),and up-regulated in the shHoxa9 group(P<0.05).In summary,Hoxa9 silencing may promote osteogenic differentiation of bone marrow mesenchymal stem cells and thereby promote fracture healing.

关 键 词：HOXA9 成骨分化 胫骨骨折 骨折愈合 细胞增殖 骨髓间充质干细胞 成骨细胞 成骨分化 动物模型 

分 类 号：R453[医药卫生—治疗学] R36[医药卫生—临床医学]