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中华口腔医学研究杂志(电子版) ›› 2021, Vol. 15 ›› Issue (04) : 207 -214. doi: 10.3877/cma.j.issn.1674-1366.2021.04.003

基础研究

骨髓间充质干细胞来源的外泌体促进髁突软骨细胞再生的研究
邢超1, 徐灵巧1, 廖文婷2, 孙养鹏2, 叶钟泰1, 张志光2,()   
  1. 1. 深圳市宝安区人民医院口腔科 518101
    2. 中山大学附属口腔医院,光华口腔医学院,广东省口腔医学重点实验室,广州 510055
  • 收稿日期:2021-04-06 出版日期:2021-08-01
  • 通信作者: 张志光

Condyle chondrocytes regeneration promoted by exosomes from bone marrow mesenchymal stem cells

Chao Xing1, Lingqiao Xu1, Wenting Liao2, Yangpeng Sun2, Zhongtai Ye1, Zhiguang Zhang2,()   

  1. 1. Dpartment of Stomatology, People′s Hospital of Shenzhen Baoan District, Shenzhen 518101, China
    2. Hospital of Stomatology, Guanghua School of Stomatology, Sun Yat-sen University, Guangdong Provincal Key Laboratory of Stomatology, Guangzhou 510055, China
  • Received:2021-04-06 Published:2021-08-01
  • Corresponding author: Zhiguang Zhang
  • Supported by:
    Basic Research of Medical and Health Care Project of Bao′an District, Shenzhen(2020JD351)
引用本文:

邢超, 徐灵巧, 廖文婷, 孙养鹏, 叶钟泰, 张志光. 骨髓间充质干细胞来源的外泌体促进髁突软骨细胞再生的研究[J/OL]. 中华口腔医学研究杂志(电子版), 2021, 15(04): 207-214.

Chao Xing, Lingqiao Xu, Wenting Liao, Yangpeng Sun, Zhongtai Ye, Zhiguang Zhang. Condyle chondrocytes regeneration promoted by exosomes from bone marrow mesenchymal stem cells[J/OL]. Chinese Journal of Stomatological Research(Electronic Edition), 2021, 15(04): 207-214.

目的

探讨大鼠骨髓间充质干细胞(BMSC)外泌体(Exo)对髁突软骨细胞的细胞增殖、细胞周期及相关基因表达的影响。

方法

获取大鼠BMSC和髁突软骨细胞。收集第三代BMSC培养72 h上清液,采用超速离心法提取Exo。实验组使用外泌体(BMSC-Exo)进行髁突软骨细胞培养,对照组使用低糖培养基进行髁突软骨细胞培养。细胞计数(CCK-8)法检测髁突软骨细胞的增殖情况,流式细胞仪分析细胞周期改变情况,反转录聚合酶链反应(RT-PCR)分析第7天及第14天髁突软骨细胞Ⅰ型胶原(ColⅠ)、Ⅱ型胶原(ColⅡ)基因表达。采用独立样本t检验进行统计学分析。

结果

CCK-8结果显示,培养72 h后实验组细胞增殖活力(4.72 ± 0.58)高于对照组(2.91 ± 0.17),差异有统计学意义(t = 16.13,P<0.001);流式细胞仪显示,Exo促进髁突软骨细胞G1期转换为G2期;RT-PCR结果显示:第7天实验组ColⅠ表达增量(2.85 ± 0.14)高于对照组(1.00 ± 0.17),差异有统计学意义(t = 13.41,P<0.001),第14天实验组ColⅠ表达增量(5.34 ± 0.17)高于对照组(3.14 ± 0.10),差异有统计学意义(t = 14.25,P<0.001),第14天实验组ColⅡ表达增量(1.77 ± 0.13)高于对照组(1.35 ± 0.18),差异有统计学意义(t = 3.52,P = 0.041)。

结论

大鼠骨髓间充质干细胞外泌体可以促进髁突软骨细胞增殖,还能促进髁突软骨细胞的ColⅠColⅡ表达。

Objective

The aim of this study was to determine the effects of exosomes from rat bone marrow mesenchymal stem cells (BMSCs) on condyle chondrocytes.

Methods

Exosomes (Exos) was extracted from rat BMSCs to culture rat condyle chondrocytes. Cell counting kit-8 (CCK-8) was applied to analyze cell proliferation of condyle chondrocytes of BMSC-Exos group and control group (L-DMEM) . Cell cycle progression was analyzed by flow cytometry. RT-PCR was applied to measure chondrogenic gene expression levels[Collagen I (Col I) and Col II) ]. Comparisons between groups were performed with independent-samples T test.

Results

The results of CCK-8 showed that the cell proliferation activity of BMSC-Exos group was 4.72 ± 0.58, which was significantly higher than that of the control group, 2.91 ± 0.17 (t = 16.13, P<0.001) . The results of flow cytometry showed that Exos could promote condyle chondrocytes transformed from G1 to G2. The results of RT-PCR indicated that the expression increment of type Col I gene in BMSC-Exos group at day 7 was 2.85 ± 0.14, which was higher than that in the control group 1.00 ± 0.17 (t = 13.41, P<0.001) . Besides, the increment of expression of Col I gene in BMSC-Exos group I on day 14 was 5.34 ± 0.17, which was higher than that in the control group 3.14 ± 0.10 (t = 14.25, P<0.001) . Meanwhile, the increment of Col II gene expression in the experimental group was 1.77 ± 0.13, which was higher than that in the control group 1.35 ± 0.18 at the 14th day (t = 3.52, P = 0.041) .

Conclusions

Exosomes from rat bone marrow mesenchymal stem cells can promote the condyle chondrocytes proliferation. Besides, it can upregulate the expression of Col I and Col II in condyle chondrocytes.

表1 反转录聚合酶链反应(RT-PCR)引物序列
图1 大鼠骨髓间充质干细胞(BMSC)形态学观察及三向分化诱导 A:显微镜下BMSC形态观察(低倍放大);B:成骨诱导21 d形成钙化结节(茜素红 低倍放大);C:成脂诱导21 d大量脂滴分散在细胞周围(苏丹黑 低倍放大);D:成软骨诱导21 d形成乳白色块状软骨
图2 大鼠髁突软骨细胞形态学观察及Ⅱ型胶原(ColⅡ)观测 A:大鼠髁突软骨细胞(低倍放大);B:DAPI细胞核染色(中倍放大);C:胞质内ColⅡ免疫荧光(中倍放大);D:DAPI细胞核染色+胞质内ColⅡ表达(中倍放大)
图3 大鼠骨髓间充质干细胞外泌体的鉴定 A:透射电子显微镜下外泌体形态;B:纳米颗粒追踪分析;C:Western blot检测特异性标记蛋白
图4 细胞计数(CCK-8)检测SD大鼠髁突软骨细胞吸光度值 实验组与对照组相比差异有统计学意义(aP<0.001)
图5 大鼠髁突软骨细胞周期分析 A~C:实验组流式细胞仪检测;D~F:对照组流式细胞仪检测;G:实验组细胞周期分析;H:对照组细胞周期分析;I:细胞周期检测结果统计图,实验组与对照组相比,差异有统计学意义(aP<0.05,bP<0.001)
图6 反转录聚合酶链反应(RT-PCR)检测大鼠髁突软骨细胞相关基因表达水平 A:Ⅰ型胶原(ColⅠ);B:Ⅱ型胶原(ColⅡ);实验组与对照组相比,差异有统计学意义(aP<0.001,bP<0.05)
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