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2型糖尿病对大鼠颌骨骨髓间充质干细胞生物学特性的影响及其机制研究

李天琪 孟祥博 霍娜 蔡川 李帅臣 周孙欣 张彤

李天琪, 孟祥博, 霍娜, 蔡川, 李帅臣, 周孙欣, 张彤. 2型糖尿病对大鼠颌骨骨髓间充质干细胞生物学特性的影响及其机制研究[J]. 解放军医学院学报, 2023, 44(4): 372-379, 387. doi: 10.3969/j.issn.2095-5227.2023.04.010
引用本文: 李天琪, 孟祥博, 霍娜, 蔡川, 李帅臣, 周孙欣, 张彤. 2型糖尿病对大鼠颌骨骨髓间充质干细胞生物学特性的影响及其机制研究[J]. 解放军医学院学报, 2023, 44(4): 372-379, 387. doi: 10.3969/j.issn.2095-5227.2023.04.010
LI Tianqi, MENG Xiangbo, HUO Na, CAI Chuan, LI Shuaichen, ZHOU Sunxin, ZHANG Tong. Effect of type 2 diabetes mellitus on biological characteristics of jaw bone marrow mesenchymal stem cells in rats and its related mechanisms[J]. ACADEMIC JOURNAL OF CHINESE PLA MEDICAL SCHOOL, 2023, 44(4): 372-379, 387. doi: 10.3969/j.issn.2095-5227.2023.04.010
Citation: LI Tianqi, MENG Xiangbo, HUO Na, CAI Chuan, LI Shuaichen, ZHOU Sunxin, ZHANG Tong. Effect of type 2 diabetes mellitus on biological characteristics of jaw bone marrow mesenchymal stem cells in rats and its related mechanisms[J]. ACADEMIC JOURNAL OF CHINESE PLA MEDICAL SCHOOL, 2023, 44(4): 372-379, 387. doi: 10.3969/j.issn.2095-5227.2023.04.010

2型糖尿病对大鼠颌骨骨髓间充质干细胞生物学特性的影响及其机制研究

doi: 10.3969/j.issn.2095-5227.2023.04.010
基金项目: 军委后勤保障部保健专项课题(19BJZ22);军队医学科技青年培育项目(21QNPY104)
详细信息
    作者简介:

    李天琪,女,在读硕士。研究方向:口腔医学。Email: 619028753@qq.com

    通讯作者:

    张彤,女,博士,主任医师。Email: kqzhengji301@163.com

  • 中图分类号: R587.2

Effect of type 2 diabetes mellitus on biological characteristics of jaw bone marrow mesenchymal stem cells in rats and its related mechanisms

More Information
  • 摘要:   背景  2型糖尿病患者骨代谢异常,颌骨骨质和骨量都发生明显改变,是牙周炎的全身促进因素之一。Wnt信号通路可以促进干细胞矿化并修复骨组织缺损。然而2型糖尿病影响下Wnt信号通路是否参与了对颌骨骨髓间充质干细胞(jaw bone marrow mesenchymal stem cells,JBMMSCs)的成骨分化调控仍未可知。  目的  研究2型糖尿病对大鼠JBMMSCs增殖及分化等生物学特性的影响,并对其机制进行初步探索。  方法  选取连续两周随机血糖≥16.7mmoL/L的13周龄GK大鼠为2型糖尿病组,相同周龄的Wistar大鼠作为对照组,两组各10只;无菌条件下采用骨髓冲洗法与骨片消化法相结合的方法分离培养两组JBMMSCs作为研究对象。CCK-8法检测并比较两组细胞增殖能力,流式细胞术检测细胞被诱导凋亡的能力。对JBMMSCs进行成骨、成脂诱导后,qRT-PCR评估成骨、成脂及Wnt信号通路相关基因的表达改变,碱性磷酸酶(alkaline phosphatase,ALP)染色检测成骨诱导后ALP的表达变化,茜素红染色比较钙结节形成能力,油红O染色检测成脂诱导后脂滴形成能力。  结果  与对照组相比,2型糖尿病组JBMMSCs的增殖及克隆形成能力降低,凋亡早期与晚期的细胞比例增加(P<0.05)。两组JBMMSCs成骨诱导后成骨相关基因ALP、OCN和 Runx2 mRNA表达升高,但2型糖尿病组低于对照组(P<0.05),2型糖尿病组钙结节形成能力和ALP染色面积及密度较对照组低(P<0.05)。成脂诱导后,成脂相关基因的表达和脂滴的形成较对照组减少(P<0.05)。2型糖尿病组JBMMSCs成骨诱导7 d后Wnt信号通路相关分子Wnt4、Wnt5a、Wnt7b的mRNA表达较对照组升高,β-catenin的表达水平较对照组低。  结论  2型糖尿病影响大鼠颌骨骨髓间充质干细胞的增殖和克隆、抑制成骨及成脂分化能力,其成骨能力的降低可能与Wnt信号通路相关。

     

  • 图  1  对照组和2型糖尿病组随机血糖和体质量

    Figure  1.  Randomized blood glucose and weight in control and T2DM groups

    图  2  对照组和2型糖尿病组JBMMSCs镜下形态观察

    A:原代培养第3天;B:原代培养第5天;C:第1代(标尺=100 µm)

    Figure  2.  Morphological observation of JBMMSCs in control and T2DM groups (bar=100 µm)

    A: P0 passage on day3; B:P0 passage on day5; C: P1 passage

    图  3  2型糖尿病对JBMMSCs增殖、克隆及凋亡的影响

    A:CCK-8法检测细胞的增殖能力;B:细胞克隆形成能力;C:细胞凋亡检测(aP<0.05,bP<0.001,vs 2型糖尿病组)

    Figure  3.  Effect of type 2 diabetes on the proliferation, clone formation and apoptosis ability of JBMMSCs

    A: Proliferation ability by CCK-8; B: Clone-forming ability; C: Detection of apoptosis (aP<0.05, bP<0.001, vs T2DM group)

    图  4  两组JBMMSCs成骨分化能力比较

    A:qRT-PCR检测正常培养和成骨诱导7 d后对照组和2型糖尿病组中成骨相关基因的mRNA表达水平的变化(aP<0.05,vs 对照组-未成骨诱导;bP<0.05,vs 对照组-成骨诱导;cP<0.05,vs 2型糖尿病组-未成骨诱导);B:ALP染色;C:茜素红染色

    Figure  4.  Comparison of osteogenic differentiation ability of JBMMSCs between the two groups

    A: Changes in mRNA expression levels of osteogenesis-related genes in the control group and the T2DM group after 7 days of normal culture and osteogenesis induction by qRT-PCR (aP<0.05, vs control-NC group; bP<0.05, vs control-OS group; cP<0.05, vs T2DM-NC group); B: ALP staining; C: Alizarin red staining

    图  5  两组JBMMSCs成脂分化能力比较

    A:qRT-PCR检测正常培养和成脂诱导7 d后对照组和2型糖尿病组中成骨相关基因的mRNA表达水平的变化(aP<0.05,vs 对照组-未成脂诱导;bP<0.05,vs 对照组-成脂诱导;cP<0.05,vs 2型糖尿病组-未成脂诱导);B:油红O染色(标尺=100 µm)

    Figure  5.  Comparison of adipogenic differentiation ability of JBMMSCs between the two groups

    A: Changes in mRNA expression levels of adipogenesis-related genes in the control group and the T2DM group after 7 days of normal culture and adipogenic differentiation induction by qRT-PCR (aP<0.05, vs control-NC group; bP<0.05, vs control-AD group; cP<0.05, vs T2MD-NC group); B: Oil Red O staining (bar=100 µm)

    图  6  Wnt信号通路相关分子mRNA水平在两组JBMMSCs成骨诱导过程中的变化(aP<0.05,vs 对照组-未成骨诱导;bP<0.05,vs对照组-成骨诱导;cP<0.05,vs 2型糖尿病组-未成骨诱导)

    Figure  6.  Changes in mRNA levels of Wnt signaling pathway-related molecules during osteogenesis induction (aP<0.05, vs control-NC group; bP<0.05, vs control-OS group; cP<0.05, vs T2DM-NC group)

    表  1  相关基因引物序列

    Table  1.   Primer sequences of related genes

     基因序列 (5'-3')
    GAPDHF: ACCCAGAAGACTGTGGATGG
    R: CACATTGGGGGTAGGAACAC
    ALPF:CACGTTGACTGTGGTTACTGCTGA
    R:CCTTGTAACCAGGCCCGTTG
    OCNF:GGTGGTGAATAGACTCCGGC
    R:GCAACACATGCCCTAAACGG
    Runx2F:GCACCCAGCCCATAATAGA
    R:TTGGAGCAAGGAGAACCC
    LPLF:GGAGTTTGGCTCCAGAGTTT
    R:AAGGTTTTGCTGCTGTGGTTG
    PPARγF:ACCGCCCAGGCTTGCTGAAC
    R:TGGAGCACCTTGGCGAACAGC
    β-cateninF:AAGTTCTTGGCTATTACGACA
    R:ACAGCACCTTCAGCACTCT
    Wnt4F: TCAGGTTGGCCACGCACTAAAGGAG
    R: AGTCTGGACTTGGCTCCAGGTACAC
    Wnt5aF:GCGGGACTTTCTCAAGGACA
    R:CGGCTGCCTATTTGCATCAC
    Wnt7bF:CTGGGAGCCAACATCATCTG
    R:TGCCCAAAGACGGTCTTCTC
    下载: 导出CSV
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  • 收稿日期:  2022-10-11
  • 网络出版日期:  2023-04-07
  • 刊出日期:  2023-04-28

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