中国组织工程研究

中国组织工程研究 ›› 2022, Vol. 26 ›› Issue (24): 3785-3789.doi: 10.12307/2022.554

• 骨髓干细胞 bone marrow stem cells • 上一篇    下一篇

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

陈巧玲1，白亦光2，刘  康3，林  涛2，罗栩伟2   

  1. 川北医学院第二临床医学院•南充市中心医院，1肿瘤科，2骨科，3组织工程与干细胞研究所，四川省南充市 637000
  • 收稿日期:2021-04-28 接受日期:2021-08-09 出版日期:2022-08-28 发布日期:2022-01-22
  • 通讯作者: 白亦光，博士，主治医师，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
  • 作者简介:陈巧玲，女，1983年生，博士，副主任医师，主要从事肿瘤学基础及临床研究。
  • 基金资助:
    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

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

Chen Qiaoling1, Bai Yiguang2, Liu Kang3, Lin Tao2, Luo Xuwei2   

  1. 1Department of Oncology, 2Department of Orthopedics, 3Institute of Tissue Engineering and Stem Cells, Second Clinical Institute of North Sichuan Medical College• Nanchong Central Hospital, Nanchong 637000, Sichuan Province, China
  • Received:2021-04-28 Accepted:2021-08-09 Online:2022-08-28 Published:2022-01-22
  • Contact: Bai Yiguang, MD, Attending physician, Department of Orthopedics, Second Clinical Institute of North Sichuan Medical College• Nanchong Central Hospital, Nanchong 637000, Sichuan Province, China
  • About author:Chen Qiaoling, MD, Associate chief physician, Department of Oncology, Second Clinical Institute of North Sichuan Medical College• Nanchong Central Hospital, Nanchong 637000, Sichuan Province, China
  • Supported by:
    Project of Sichuan Health and Family Planning Commission, No. 16PJ202 (to BYG); Nanchong University Science and Technology Strategic Cooperation Project, No. 18SXHZ0539 (to BYG)

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摘要：

文题释义：
3-磷酸肌醇依赖性蛋白激酶 1(PDK-1)：作为 AGC 蛋白的上游分子可通过依赖磷脂酰肌醇3，4，5-三磷酸性方式激活蛋白激酶 B(AKT)苏氨酸残基308。
Cre重组酶技术：Cre是来源于噬菌体P1的一种酶蛋白。它可以识别催化两个LoxP位点之间发生同源重组，从而造成DNA的缺失、易位等现象。

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-1flox/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-1flox/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。
缩略语：3-磷酸肌醇依赖的蛋白激酶1：3-phosphoinositide-dependent protein kinase-1，PDK-1

https://orcid.org/0000-0002-5656-6199(白亦光) 

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

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

Abstract: 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-1flox/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-1flox/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. 

Key words: metastatic bone tumor, osteoporosis, bone destruction, osteoblasts, PDK-1, signaling pathway

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