中文摘要：

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：

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, KRTI9 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-1α and KRT19 mRNA expressions 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.