髓核细胞来源外泌体对骨髓间充质干细胞向髓核样细胞分化的作用

Exosomes derived from nucleus pulposus cells induced bone marrow mesenchymal stem cells into nucleus pulposus like cells

中文关键词:  椎间盘退变  外泌体  髓核细胞  骨髓间充质干细胞

英文关键词:Intervetebral disc degeneration  Exosomes  Mesenchymal stem cells  Nucleus pulposus cells

中文摘要:

  【摘要】 目的：探讨人髓核细胞（NPCs）外泌体对人骨髓间充质干细胞（BMSCs）向髓核样细胞分化的作用。方法：取腰椎间盘突出症患者手术切除的髓核组织，体外分离培养人NPCs，采用差速离心法提取NPCs的外泌体，利用透射电镜及Western blot对外泌体进行大小形态及标志蛋白的检测，同时用PKH67荧光染料标记外泌体后与BMSCs共孵育0.5h、2h、4h，在激光共聚焦显微镜下观察BMSCs对NPCs外泌体的摄取情况；取人BMSCs经NPCs外泌体诱导3d、7d、10d、14d后应用RT-PCR检测BMSCs中蛋白聚糖（ACAN）、SOX-9、Ⅱ型胶原（COL2A1）、角蛋白19（KRT19）及缺氧诱导因子1α（HIF-1α）的mRNA表达情况。结果：提取的人NPCs外泌体为直径为30~100nm的圆形或椭圆形结构，其表达CD63和Tsg101，不表达Calnexin蛋白。经PKH67标记的NPCs外泌体可以被BMSCs摄取；经NPCs外泌体诱导后7d开始BMSCs中的ACAN、SOX-9、COL2A1、KRT19及HIF-1α基因mRNA表达均显著性高于未经诱导的BMSCs（P<0.05）。结论：在体外实验中，人NPCs可以分泌外泌体并被BMSCs所摄取，诱导BMSCs分化为髓核样细胞，可为椎间盘退变的组织工程修复提供更为简单有效的NPCs来源。

英文摘要:

  【Abstract】 Objectives: To investigate the role of exosomes derived from nucleus pulposus cells(NPCs) in inducing bone marrow mesenchymal stem cells(BMSCs) differentiation into NP-like cells. Methods: The nucleus pulposus was obtained from the surgical specimen of the patient diagnosed as lumbar disc herniation. The NPCs were separated and cultured in vitro, and then the NPC-exosomes were isolated and purified by differential centrifugation. NPC-exosomes were identified by transmission electron microscope(TEM) and immuneblot analysis. To test the uptake of NPC-exosomes by BMSCs, PKH67 labeled NPC-exosomes were co-incubated with BMSCs for 0.5, 2, 4 hours. Fluorescence confocal microscope(FCFM) was used to examine the uptake of NPC-exosomes. After 3, 7, 10, 14 days stimulated by NPC-exosomes, RT-PCR analysis was performed to detect the mRNA expressions of ACAN, SOX9, COL2A1, KRT19 and HIF-1α in BMSCs. Results: Exosomes derived from NPCs were observed on TEM and the size of them was in the range of 30-100nm. Additionally, NPC-exosomes expressed the exosomal markers CD63, TSG101 and no endoplasmic reticulum marker Calnexin. FCM confirmed that NPCs derived exosomes could be transferred into BMSCs. The RT-PCR results showed that with the duration of interaction, ACAN, SOX-9, COL2A1, HIF-1 α and KRT19 mRNA expres?鄄sions in BM-MSCs had significant increases compared with those of the control group(P<0.05). Conclusions: NPCs can secrete exosomes and then interact with BMSCs to induce BMSCs differentiation into NP-like cells. This study provides a more efficient and simple source of NPCs for tissue engineering repair of degenerative intervertebral disc.