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中华口腔医学研究杂志(电子版)

中华口腔医学研究杂志(电子版) ›› 2018, Vol. 12 ›› Issue (02) : 101 -109. doi: 10.3877/cma.j.issn.1674-1366.2018.02.006

所属专题： 文献；

基础研究

脂肪间充质干细胞条件培养基及其外泌体促成骨作用的体外研究
黄春煌1, 马媛媛1, 任林1, 蔡庆伟1, 陈瑞欣1, 付强1,†()   
  1. 1. 510055　广州，中山大学光华口腔医学院·附属口腔医院，广东省口腔医学重点实验室
  • 收稿日期:2017-12-19 出版日期:2018-04-01
  • 通信作者: 付强
  • 基金资助:
    国家自然科学基金(81570955); 广东省科技计划(2013B051000032、2014A020212072); 广州市科技计划(201510010222)

Conditioned media and exosomes from rat adipose-derived mesenchymal stem cells enhance bone regeneration: a study in vitro

Chunhuang Huang1, Yuanyuan Ma1, Lin Ren1, Qingwei Cai1, Ruixin Chen1, Qiang Fu1,†()   

  1. 1. Guanghua School of Stomatology, Hospital of Stomatology, Sun Yat-sen University, Guangdong Provincial Key Laboratory of Stomatology, Guangzhou 510055, China
  • Received:2017-12-19 Published:2018-04-01
  • Corresponding author: Qiang Fu
  • About author:
    Corresponding author:Fu Qiang,Email:
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黄春煌, 马媛媛, 任林, 蔡庆伟, 陈瑞欣, 付强. 脂肪间充质干细胞条件培养基及其外泌体促成骨作用的体外研究[J]. 中华口腔医学研究杂志(电子版), 2018, 12(02): 101-109.

Chunhuang Huang, Yuanyuan Ma, Lin Ren, Qingwei Cai, Ruixin Chen, Qiang Fu. Conditioned media and exosomes from rat adipose-derived mesenchymal stem cells enhance bone regeneration: a study in vitro[J]. Chinese Journal of Stomatological Research(Electronic Edition), 2018, 12(02): 101-109.

目的

研究大鼠脂肪间充质干细胞（ASC）条件培养基及其外泌体的体外促成骨作用。

方法

分离培养原代大鼠ASC和骨髓间充质干细胞（BMSC）并进行鉴定；超速离心法收集/去除条件培养基中的外泌体，采用透射电镜法及粒径分析法鉴定所获取的外泌体；细胞计数试剂盒（CCK-8）法检测其对细胞增殖的影响；Transwell检测其对细胞迁移的影响；碱性磷酸酶（ALP）活性实验和实时荧光定量聚合酶链反应（PCR）检测其对成骨的影响。SPSS 20.0软件、Bonferroni检验进行统计学分析。

结果

实验所获取的原代BMSC和ASC符合间充质干细胞鉴定标准。透射电镜及粒径分析结果表明所获取的外泌体具有典型的外泌体形态特征。CCK-8实验表明培养7 d后，各处理组中A450值存在差异（F= 157.74，P= 0.001），BMSC增殖条件培养基组A450值（2.71 ± 0.15）高于对照组（1.95 ± 0.11）；去外泌体组的A450值（2.28 ± 0.34）低于条件培养基组。Transwell实验结果表明，培养24 h后各处理组中的迁移细胞数存在差异（F= 46.79，P= 0.001），条件培养基组迁移细胞数（51.6 ± 1.3）高于对照组（28.6 ± 1.4），去外泌体组迁移的细胞数（37.2 ± 2.2）低于条件培养基组。ALP活性实验结果表明，培养7 d后各处理组中的ALP活性存在差异（F= 78.43，P= 0.001），条件培养基组ALP活性为（310 ± 11），高于对照组（200 ± 24），去外泌体组ALP活性为（240 ± 19）低于条件培养基组。实时荧光定量PCR检测成骨相关基因的表达，各处理组中的基因表达存在差异（FALP= 32.30，PALP= 0.001；FRUNX2= 26.78，PRUNX2= 0.001）。

结论

成骨诱导后大鼠ASC条件培养基及其中的外泌体能够有效的促进BMSC增殖、迁移和成骨向分化，具有体外促成骨作用。

关键词: 间质干细胞, 外泌体, 培养基，条件性, 骨和骨组织，再生
Objective

To investigate the effect of conditioned media and exosomes from rat adipose-derived mesenchymal stem cells (ASCs) on bone regeneration in vitro.

Methods

Primary ASCs and bone marrow-derived mesenchymal stem cells (BMSCs) were isolated, cultured and identified. ASC-derived exosomes were isolated/depleted by ultracentrifugation and identified by transmission electron microscope and particle size analysis. CCK-8 and Transwell assays were used to evaluate cell proliferation and migration, respectively. The osteogenic differentiation in BMSCs were evaluated by qRT-PCR and ALP activity test. The mean values between groups were analyzed by Bonferroni Test in SPSS 20.0.

Results

The ASCs and BMSCs were confirmed. Transmission electron microscope analysis and particle size analysis showed ASC-derived exosomes after osteoinduction with characteristic morphology and size. CCK-8 assay showed significant difference among groups after 7 d culture (F= 157.74, P= 0.001) . Cell proliferation in conditioned media group (A450= 2.71±0.15) was significantly higher than that in control (A450= 1.95 ± 0.11) , and exosome deleted group (A450= 2.28 ± 0.34) . Transwell assay showed significant difference among groups after 24 h culture (F= 46.79, P= 0.001) . Migrating cells in conditioned media group (51.6 ± 1.3) was significantly higher than that in control (28.6±1.4) and exosome deleted group (37.2 ± 2.2) . ALP activity assay showed significant difference among groups after 7 d culture (F= 78.43, P= 0.001) . ALP content in conditioned media group (310 ± 11) was significantly higher than that in control (200 ± 24) and exosome deleted group (125 ± 15) . QPCR showed significant difference among groups (FALP= 32.30, PALP= 0.001; FRUNX2= 26.78, PRUNX2= 0.001) .

Conclusion

ASC-CM and exosomes can enhance the proliferation, migration, and osteogenic differentiation of BMSCs and promote bone regeneration in vitro.

Key words: Mesenchymal stem cells, Exosomes, Culture media, conditioned, Bone and bones, regeneration
表1 实时荧光定量PCR引物序列
基因 引物序列
RUNX2 F:5′-TTCAAGGTTGTAGCCCTCGGAG-3′
? R:5′-TCAAAGTGAAACTCTTGCCTCGT-3′
ALP F:5′-AGAACAGAACTGATGTGGAATATGAA-3′
? R:5′-CAGTGCGGTTCCAGACATAGT-3′
β-actin F:5′-AGCCATGTACGTAGCCATCCA-3′
? R:5′-TCTCCGGAGTCCATCACAATG-3′
图1 大鼠骨髓间充质干细胞（BMSC）和脂肪间充质干细胞（ASC）形态及克隆形成鉴定（低倍放大）
图2 大鼠骨髓间充质干细胞（BMSC）和脂肪间充质干细胞（ASC）多向分化能力鉴定
图3 大鼠骨髓间充质干细胞（BMSC）和脂肪间充质干细胞（ASC）表面标志物流式鉴定
图4 大鼠脂肪间充质干细胞（ASC）外泌体透射电镜下形态
图5 大鼠脂肪间充质干细胞（ASC）外泌体粒径分析
表2 不同处理组作用对BMSC增殖活性检测结果（A450， ± s）
组别 1d 3d 5d 7d
对照组 0.94 ± 0.11 1.35 ± 0.16 1.72 ± 0.07 1.95 ± 0.11
条件培养基组 1.03 ± 0.08 2.00 ± 0.15 2.47 ± 0.21 2.71 ± 0.15
去外泌体组 0.98 ± 0.12 1.56 ± 0.10 2.05 ± 0.11 2.28 ± 0.34
F值 2.87 86.71 106.09 157.74
P值 0.068 0.001 0.001 0.001
图6 不同处理组对骨髓间充质干细胞（BMSC）增殖的影响
图7 不同处理组对骨髓间充质干细胞（BMSC）迁移的影响
图8 不同处理组对骨髓间充质干细胞（BMSC）碱性磷酸酶（ALP）活性的影响
图9 不同处理组对骨髓间充质干细胞（BMSC）成骨相关mRNA表达的影响
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