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miR-145-5p在正常和2型糖尿病大鼠骨髓间充质干细胞成骨分化中的表达比较

戴雅文 鄂玲玲 郑颖 马小草 张戎 时权 刘洪臣

戴雅文, 鄂玲玲, 郑颖, 马小草, 张戎, 时权, 刘洪臣. miR-145-5p在正常和2型糖尿病大鼠骨髓间充质干细胞成骨分化中的表达比较[J]. 解放军医学院学报.
引用本文: 戴雅文, 鄂玲玲, 郑颖, 马小草, 张戎, 时权, 刘洪臣. miR-145-5p在正常和2型糖尿病大鼠骨髓间充质干细胞成骨分化中的表达比较[J]. 解放军医学院学报.
DAI Yawen, E Lingling, ZHENG Ying, MA Xiaocao, ZHANG Rong, SHI Quan, LIU Hongchen. Comparison of expression of miR-145-5p in osteogenic differentiation of bone marrow mesenchymal stem cells derived from normal versus type 2 diabetic rats[J]. ACADEMIC JOURNAL OF CHINESE PLA MEDICAL SCHOOL.
Citation: DAI Yawen, E Lingling, ZHENG Ying, MA Xiaocao, ZHANG Rong, SHI Quan, LIU Hongchen. Comparison of expression of miR-145-5p in osteogenic differentiation of bone marrow mesenchymal stem cells derived from normal versus type 2 diabetic rats[J]. ACADEMIC JOURNAL OF CHINESE PLA MEDICAL SCHOOL.

miR-145-5p在正常和2型糖尿病大鼠骨髓间充质干细胞成骨分化中的表达比较

基金项目: 国家自然科学基金项目(82170985,81930026)
详细信息
    作者简介:

    戴雅文,女,在读硕士,医师。研究方向:干细胞与成骨分化。Email:395298305@qq.com

    通讯作者:

    刘洪臣,男,博士,主任医师,主任。Email:liu-hc301@hotmail.com

  • 中图分类号: R587.1

Comparison of expression of miR-145-5p in osteogenic differentiation of bone marrow mesenchymal stem cells derived from normal versus type 2 diabetic rats

More Information
  • 摘要:   背景  糖尿病导致骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMMSCs) 成骨分化能力降低, miRNAs在此过程中发挥重要作用,其中miR-145-5p对细胞成骨成软骨分化有重要的调节作用。然而miR-145-5p对糖尿病源BMMSCs成骨分化的影响尚不清楚。  目的  比较正常和2型糖尿病大鼠BMMSCs成骨分化能力,初步探讨在成骨分化中miR-145-5p及其靶基因SEMA3A和Wnt通路关键蛋白β-catenin表达的差异。  方法  12只GK大鼠采用高糖高脂饲料喂养构建2型糖尿病大鼠模型,12只Wistar大鼠常规饲养作为对照组。无菌条件下分离大鼠股骨,全骨髓培养法培养正常和2型糖尿病大鼠BMMSCs(分别对应WT-BMMSCs和GK-BMMSCs);CCK-8检测细胞增殖能力;结晶紫染色检测细胞集落形成能力;碱性磷酸酶染色及半定量分析检测碱性磷酸酶活性;茜素红染色检测矿化基质形成;qRT-PCR和Western blot检测成骨相关标志物、miRNA-145-5p、SEMA3A和β-catenin的表达。  结果  干细胞培养4-9 d时,GK-BMMSCs的增殖低于WT-BMMSCs,差异有统计学意义(P<0.01);10 d时,其集落形成率显著低于WT-BMMSCs(P<0.01);成骨诱导7 d时,GK-BMMSCs碱性磷酸酶(alkaline phosphatase,ALP)活性(P<0.001)、成骨相关标志物碱性磷酸酶基因 (P<0.01)和蛋白(P<0.001)表达、骨钙素(osteocalcin,OCN) 基因(P<0.001)和蛋白(P<0.001)表达均显著低于WT-BMMSCs,1型胶原蛋白(type 1 collagen,COL1)(P<0.01)基因的表达显著低于WT-BMMSCs,而Runt相关转录因子2(Runt-related transcription factor-2,Runx2)(P<0.001)蛋白表达显著高于WT-BMMSCs。在WT-BMMSCs成骨分化中,miR-145-5p表达下调,SEMA3A表达上调,而在GK-BMMSCs成骨分化中,miR-145-5p和SEMA3A表达均上调,β-catenin(P<0.001)在GK-BMMSCs中的表达显著降低。成骨诱导21 d时,WT-BMMSCs矿化基质染色较深。  结论  2型糖尿病大鼠BMMSCs细胞增殖、集落形成及成骨分化能力降低,推测在正常大鼠BMMSCs成骨分化中miR-145-5p起抑制作用,在2型糖尿病大鼠BMMSCs成骨分化中miR-145-5p起促进作用。

     

  • 图  1  8-13周Wistar与GK大鼠体重

    (n=12) aP<0.001

    Figure  1.  Body weight of Wistar and GK rats at 8-13 weeks (n=12) aP<0.001

    图  2  8-13周Wistar与GK大鼠血糖

    (n=12) aP<0.001

    Figure  2.  Blood glucose of Wistar and GK rats at 8-13 weeks (n=12) aP<0.001

    图  3  倒置显微镜观察原代(p0)与第三代(p3)正常和糖尿病大鼠BMMSCs (标尺 100 μm)

    Figure  3.  Primary and the third generation BMMSCs derived from normal and type 2 diabetic rats (scale bar=100 μm)

    图  4  流式细胞学鉴定BMMSCs表面抗原

    Figure  4.  Identification of BMMSCs surface antigen by flow cytometry

    图  5  CCK8试剂盒检测正常和糖尿病大鼠股骨BMMSCs生长曲线(n=6) aP<0.01

    Figure  5.  Detection of growth curve of BMMSCs derived from normal and type 2 diabetic rats by CCK8 kit (n=6) aP<0.01

    图  6  正常和糖尿病大鼠股骨BMMSCs集落形成实验

    Figure  6.  Colony formation experiment of femoral BMMSCs in normal rats and type 2 diabetic rats

    图  7  正常与2型糖尿病大鼠BMMSCs成骨分化能力比较

    A:成骨诱导7 d碱性磷酸酶染色(标尺 100 μm);B:成骨诱导7d ALP半定量分析 (n=6);C:成骨诱导21天茜素红染色显示钙化结节形成(标尺 100 μm)

    Figure  7.  Comparison of osteogenic differentiation capacity of BMMSCs derived from normal rats and type 2 diabetic rats

    A: ALP staining was performed on day 7 after osteogenic differentiation(scale bar=100μm); B: Quantification of ALP activity was shown(n=6); C:ARS staining was performed on day 21 after osteogenic differentiation to show the formation of calcified nodules(scale bar=100μm)

    图  8  成骨诱导7d正常与2型糖尿病大鼠BMMSCs ALP、OCN 、RUNX2、COL1基因表达(n=3)

    Figure  8.  Expression of the osteoblast differentiation-related genes ALP, OCN, RUNX2, COL1 on day 7 after osteogenic differentiation by real-time PCR (n=3)

    图  9  成骨诱导7d正常与2型糖尿病大鼠BMMSCs ALP、OCN、RUNX2蛋白水平的表达

    A: ALP、OCN、RUNX2蛋白的表达; B:ALP、OCN 、RUNX2蛋白水平表达的定量分析(n=3)

    Figure  9.  Western blot analysis of ALP, OCN, RUNX2 on day 7 after osteogenic differentiation

    A:Western blotting of ALP, OCN, RUNX2;B:Protein levels of ALP, OCN, RUNX2 quantified by densitometry(n=3)

    图  10  成骨诱导7d正常与2型糖尿病大鼠BMMSCs miR-145-5p、SEMA3A、β-catenin的表达(n=3)

    Figure  10.  Expression of miR-145-5p, SEMA3A, β-catenin on day 7 after osteogenic differentiation by real-time PCR (n=3)

    图  11  成骨诱导7天正常与2型糖尿病大鼠BMMSCs SEMA3A的蛋白表达

    A:SEMA3A蛋白的表达 B:SEMA3A蛋白水平表达的定量分析(n=3)

    Figure  11.  Western blot analysis of SEMA3A on day 7 after osteogenic differentiation

    A: Western blotting of SEMA3A; B: Protein levels of SEMA3A quantified by densitometry(n=3)

    表  1  引物序列

    Table  1.   Primer sequences

    基因 引物序列
    SEMA3A F-CTTGCTCGGGACCCTTATTG
    R-AGGCTCTCTGTGACTTCGGACT
    β-catenin F-TGCCATCTGTGCTCTTCGTC
    R-CAATCCAACAGTTGCCTTTATCAG
    ALP F-TGGTGAGTGACACGGACAAGAA
    R-GCCTGGTAGTTGTTGTGAGCAT
    COL1 F-CGTGGAAACCTGATGTATGCTTG
    R-CCTATGACTTCTGCGTCTGGTGA
    OCN F-TGACAAAGCCTTCATGTCCAA
    R-CTCCAAGTCCATTGTTGAGGTAG
    RUNX2 F-TACCCAGGCGTATTTCAGATGAT
    R-TGTAAGTGAAGGTGGCTGGATAGT
    GAPDH F-CTGGAGAAACCTGCCAAGTATG
    R-GGTGGAAGAATGGGAGTTGCT
    U6 F-CTCGCTTCGGCAGCACA
    R-AACGCTTCACGAATTTGCGT
    下载: 导出CSV
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  • 收稿日期:  2022-12-29
  • 网络出版日期:  2023-05-25

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