条件性敲除骨髓间充质干细胞中3-磷酸肌醇依赖蛋白激酶1基因后的成骨细胞分化  

Osteoblast differentiation after conditional knockout of 3-phosphoinositide-dependent protein kinase-1 gene from bone marrow mesenchymal stem cells

作　　者：陈巧玲[1] 白亦光[2] 刘康[3] 林涛[2] 罗栩伟[2] Chen Qiaoling;Bai Yiguang;Liu Kang;Lin Tao;Luo Xuwei(Department of Oncology,Second Clinical Institute of North Sichuan Medical College·Nanchong Central Hospital,Nanchong 637000,Sichuan Province,China;Department of Orthopedics,Second Clinical Institute of North Sichuan Medical College·Nanchong Central Hospital,Nanchong 637000,Sichuan Province,China;Institute of Tissue Engineering and Stem Cells,Second Clinical Institute of North Sichuan Medical College·Nanchong Central Hospital,Nanchong 637000,Sichuan Province,China)

机构地区：[1]川北医学院第二临床医学院·南充市中心医院肿瘤科,四川省南充市637000 [2]川北医学院第二临床医学院·南充市中心医院骨科,四川省南充市637000 [3]川北医学院第二临床医学院·南充市中心医院组织工程与干细胞研究所,四川省南充市637000

出　　处：《中国组织工程研究》2022年第24期3785-3789,共5页Chinese Journal of Tissue Engineering Research

基　　金：四川省卫计委课题(16PJ202),项目负责人:白亦光;南充市校科技战略合作专项(18SXHZ0539),项目负责人:白亦光。

摘　　要：背景:国内外对于3-磷酸肌醇依赖蛋白激酶1(3-phosphoinositide-dependent protein kinase-1,PDK-1)的研究主要集中在内分泌和肿瘤学等学科领域,在骨科学中关于其对成骨分化的影响尚未有系统研究与报道。目的:通过使用稳定表达cre酶的腺病毒(pHBAd-cre-EGFP)转染PDK-1^(flox/flox)小鼠骨髓间充质干细胞,观察PDK-1在成骨细胞分化中的作用。方法:体外培养获得来源于纯合子PDK-1^(flox/flox)小鼠的骨髓间充质干细胞,设立对照组、空载病毒组(pHBAd-EGFP)和pHBAd-cre-EGFP组,对照组仅用成骨细胞诱导培养基诱导培养骨髓间充质干细胞,空载病毒组使用pHBAd-EGFP转染骨髓间充质干细胞,pHBAd-cre-EGFP组使用含Cre重组酶的重组腺病毒(pHBAd-cre-EGFP)干扰骨髓间充质干细胞中的PDK-1基因,然后进行成骨诱导,采用碱性磷酸酶染色、碱性磷酸酶活性、茜素红染色和qPCR检测成骨相关基因表达来评价各组成骨细胞的分化成熟情况。结果与结论:pHBAd-cre-EGFP组细胞的碱性磷酸酶分泌、碱性磷酸酶活性、矿化能力均明显低于其他2组(P<0.05,P<0.01),成骨细胞相关基因Runx2、骨钙素和Ⅰ型胶原的表达也明显低于其他2组(P<0.01)。结果表明,体外干扰PDK-1基因表达可显著抑制骨髓间充质干细胞成骨分化。BACKGROUND:The research on 3-phosphoinositide-dependent protein kinase-1(PDK-1)in and outside China is mainly concentrated in endocrinology and oncology.There are no systematic studies and reports on its influence on osteogenic differentiation in orthopedics.OBJECTIVE:To observe the role of PDK-1 in osteoblast differentiation by transfecting PDK-1^(flox/flox) mouse bone marrow mesenchymal stem cells with adenovirus stably expressing cre enzyme(pHBAd-cre-EGFP).METHODS:Bone marrow mesenchymal stem cells from homozygous PDK-1^(flox/flox) mice were obtained and cultured in vitro.A control group,an empty virus group(pHBAd-EGFP)and a pHBAd-cre-EGFP group were set.In the control group,osteoblast induction medium was used to induce bone marrow mesenchymal stem cells.In pHBAd-EGFP group,pHBAd-EGFP was used to transfect bone marrow mesenchymal stem cells.In the pHBAd-cre-EGFP group,the recombinant adenovirus containing Cre recombinant enzyme(pHBAd-cre-EGFP)was used to transfect with the PDK-1 gene in bone marrow mesenchymal stem cells followed by osteogenic induction.Alkaline phosphatase staining,alkaline phosphatase activity assay,alizarin red staining,and qPCR were used to detect osteogenesis-related gene expression and to evaluate the differentiation and maturation of osteoblasts.RESULTS AND CONCLUSION:The alkaline phosphatase secretion,alkaline phosphatase activity,and the ability of cell mineralization in the pHBAd-cre-EGFP group were significantly lower than those in the other two groups(P<0.05,P<0.01),and the expression levels of Runx2,osteocalcin,and collagen I were also significantly lower than those in the other two groups(P<0.01).The results have confirmed that interference with PDK-1 gene in vitro can significantly inhibit the osteogenic differentiation of bone marrow mesenchymal stem cells.

关 键 词：转移性骨肿瘤 骨质疏松 骨破坏 成骨细胞 PDK-1 信号通路 

分 类 号：R459.9[医药卫生—治疗学] R318[医药卫生—临床医学]