大鼠骨髓间充质干细胞来源外泌体对退变髓核细胞的影响

Effects of exosome derived from rat bone marrow mesenchymal stem cells on degenerative nucleus pulposus cells

中文关键词:  髓核细胞  骨髓间充质干细胞  外泌体  共培养  SD大鼠

英文关键词:Nucleus pulposus cells  Bone marrow mesenchymal stem cells  Exosome  Coculture  SD rats

中文摘要:

  【摘要】 目的：探讨大鼠骨髓间充质干细胞（BMSCs）来源外泌体对退变髓核细胞（nucleus pulposus cells，NPCs）的影响。方法：利用全骨髓法提取SD大鼠骨髓内贴壁细胞，通过成骨、成脂、成软骨三系分化及流式细胞技术鉴定所提取细胞是否为BMSCs，鉴定成功后，收取细胞培养液上清，对上清液进行差速离心，Western-blot（WB）法测定离心沉淀物质CD63、CD81、TSG101、Calnexin蛋白表达情况，并对沉淀物进行透射电镜观察鉴定是否为外泌体。取SD大鼠尾NPCs，传代培养，通过细胞衰老β-半乳糖苷酶染色法鉴定P6 NPCs是否较P2 NPCs产生退变；在激光共聚焦显微镜下观察BMSCs外泌体被P6 NPCs摄取情况；设置P6 NPCs为对照组，BMSCs和P6 NPCs一起培养为共培养组，直接加入BMSCs外泌体诱导P6 NPCs为实验组。培养3d、7d、10d、14d后用RT-PCR法检测3组NPCs中蛋白聚糖（ACAN）、Ⅱ型胶原（COL2）、性别决定区Y方框9（SOX-9）、金属蛋白酶组织抑制剂1（TIMP1）、基质金属蛋白酶1（MMP1）基因mRNA的相对表达量；WB法检测ACAN、COL2、SOX-9、TIMP1、MMP1对应蛋白的相对表达情况。结果：通过成骨、成脂、成软骨三系分化及流式细胞技术鉴定利用全骨髓法提取的贴壁细胞为BMSCs。BMSCs培养液上清利用差速离心法提取的沉淀物形态为直径30～100nm的圆形或椭圆形，与文献记载的经典外泌体大小吻合，沉淀物CD63、CD81、TSG101高表达，Calnexin未表达。通过细胞衰老β-半乳糖苷酶染色法鉴定P6 NPCs较P2 NPCs产生明显退变。在激光共聚焦显微镜下观察到外泌体能够被P6 NPCs所摄取。ACAN、COL2、SOX-9、TIMP1基因mRNA的相对表达量在3d、7d、10d、14d共培养组较对照组明显升高（P＜0.05），实验组较共培养组明显升高（P＜0.05），共培养组和实验组组内7d较3d明显升高（P＜0.05）、10d较7d明显升高（P＜0.05）、14d较10d明显升高（P＜0.05）；MMP1基因mRNA的相对表达量在3d、7d、10d、14d共培养组较对照组明显降低（P＜0.05），实验组较共培养组明显降低（P＜0.05），共培养组和实验组组内7d较3d明显降低（P＜0.05）、10d较7d明显降低（P＜0.05）、14d较10d明显降低（P＜0.05）；ACAN、COL2、SOX-9、TIMP1、MMP1基因相对应蛋白质表现出同样趋势。结论：在体外实验中，大鼠BMSCs能够分泌外泌体且外泌体能够被退变NPCs摄取，外泌体能够改善退变NPCs标志基因及基因对应蛋白质的表达，可为髓核细胞退变类疾病的治疗提供一种新的思路。

英文摘要:

  【Abstract】 Objectives: To explore the effect of exosome derived from rat bone marrow mesenchymal stem cells(BMSCs) on degenerative nucleus pulposus cells(NPCs). Methods: The adherent cells were extracted from bone marrow of SD rats by whole bone marrow method. After BMSCs were successfully identified by osteoblast differentiation and flow cytometry, the supernatant of cultured cells was collected. The supernatant was centrifuged by differential centrifugation, and the expressions of CD63, CD81, TSG101, Calnexin protein in the precipitate were determined by Western-blot(WB) method, and whether the precipitate exosome or not was observed by transmission electron microscope. After extracting NPCs from SD rats tail, P6 NPCs were identified whether it was more degenerative than P2 NPCs by senescence β-galactosidase staining. The uptake of BMSCs exosome was observed by P6 NPCs under laser confocal microscope. P6 NPCs were taken as the control group, BMSCs and P6 NPCs were as the co-culture group, and P6 NPCs which were induced by BMSCs exosome were taken as the experimental group. The proteoglycan(ACAN), type Ⅱ collagen(COL2), sex-determining region Y box 9(SOX-9), tissue inhibitor of metalloproteinase 1(TIMP1) and the relative expression of matrix metalloproteinase-1(MMP1) gene mRNA of NPCs in the three groups were detected by RT-PCR method. Relative expressions of ACAN, COL2, SOX-9, TIMP1, MMP1 gene corresponding protein were detected by WB. Results: The adherent cells which were extracted by whole bone marrow method were BMSCs and they were identified by osteogenesis and adipogenic cartilage differentiation method and flow cytometry method. The precipitates extracted from BMSCs supernatant by differential centrifugation were circular or elliptical in diameter of 30-100nm, which were coincided with the classical exosome size recorded in literature. CD63, CD81, TSG101 were highly expressed and Calnexin was not expressed in the supernatant. Identified by cell senescence β-galactosidase staining, P6 NPCs were more degenerative than P2 NPCs, and the exosome could be absorbed by P6 NPCs observed under the confocal laser microscope. The relative expressions of mRNA of ACAN, COL2, SOX-9, TIMP1 gene were significantly higher in the co-culture group than those in the control group on the 3rd day, 7th day, 10th day and 14th day(P<0.05), and those in the experimental group were significantly higher than those in the co-culture group(P<0.05). In the co-culture group and the experimental group, those on the 7th day were significantly higher than those on the 3rd day(P<0.05), and those on the 10th day were significantly higher than those on the 7th day(P<0.05), and those on the 14th day were significantly higher than those on the 10th day(P<0.05). The relative expression of mRNA of MMP1 gene was significantly lower in the co-culture group than that in the control group on the 3rd day, 7th day, 10th day and 14th day(P<0.05), and that in the experimental group was significantly lower than that in the co-culture group(P<0.05). In the co-culture group and experimental group, there was a significant decrease in the 7th day compared with the 3rd day(P<0.05), and decrease also showed in the 10th day in comparison with the 7th day(P<0.05), and the 14th day in comparison with the 10th day(P<0.05). ACAN, COL2, SOX-9, TIMP1, MMP1 gene corresponding protein showed the same trend. Conclusions: In vitro, rat BMSCs can secrete exosome and the exosome can be taken up by degenerative NPCs. Exosome can improve the expression of degenerative NPCs marker gene and gene corresponding protein. It can provide a new idea for the treatment of degenerative diseases of nucleus pulposus cells.