大鼠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研究

The inducing effect of rat nucleus pulposus cells derived exosomes on the differentiation of mesenchymal stem cells into nucleus pulposus-like cells

中文关键词:  外泌体  骨髓间充质干细胞  髓核细胞  分化

英文关键词:Exosomes  Bone marrow mesenchymal stem cell  Nucleus pulposus cell  Differentiation

中文摘要:

  【摘要】 目的：探讨大鼠髓核细胞来源外泌体对大鼠骨髓间充质干细胞（BMSCs）向髓核样细胞分化的作用。方法：采用贴壁法体外分离培养SD大鼠尾椎椎间盘髓核细胞和BMSCs，流式细胞术和三系分化实验鉴定BMSCs。差速离心法分离髓核细胞外泌体，透射电镜观察其形态并使用蛋白免疫印迹（Western blot）检测其蛋白标志物CD81、Tsg101。分别使用荧光探针CM-DIO和CM-DIL标记BMSCs和髓核细胞外泌体，将两者共培养24h后在荧光显微镜下观察BMSCs对髓核细胞外泌体摄取情况。将第三代BMSCs分为三组：外泌体组，加入髓核细胞外泌体（50μg/ml）；共培养组，与髓核细胞非接触式共培养；对照组，未做任何处理。分别于7、14、21d时应用荧光定量PCR（qRT-PCR）检测外泌体组和对照组Ⅱ型胶原（COL2A1）、蛋白多糖（ACAN）、SOX9的mRNA表达量。14d时应用qRT-PCR检测3组的COL2A1、ACAN、SOX9的mRNA表达量。结果：提取的第三代大鼠髓核细胞呈多角形或不规则形状，第三代BMSCs呈形态均一的长梭形。BMSCs高表达CD29（99.77%）、CD44（93.97%）、CD90（99.67%），低表达CD34（0.82%）、CD45（0.68%）。BMSCs成骨、成脂、成软骨诱导后染色均为阳性。透射电镜观察外泌体为30~100nm类圆形双层膜结构，其表达CD81、Tsg101蛋白，不表达Calnexin蛋白。荧光显微镜下CM-DIL标记的外泌体可被CM-DIO标记的BMSCs摄取。诱导7、14、21d后，外泌体组的COL2A1、ACAN、SOX9 mRNA表达量均显著高于对照组（P＜0.05）。14d时共培养组COL2A1、ACAN、SOX9的mRNA表达量均显著高于对照组，低于外泌体组（P＜0.05）。结论：大鼠髓核细胞外泌体可在体外诱导BMSCs向髓核样细胞分化，且诱导效果优于与髓核细胞的非接触式共培养，可为椎间盘组织工程提供一种有效的髓核细胞来源。

英文摘要:

  【Abstract】 Objectives: To investigate the roles of exosomes derived from rat nucleus pulposus cells(NPCs) in the differentiation of rat bone marrow mesenchymal stem cells(BMSCs) into nucleus pulposus-like cells. Methods: SD rats were sacrificed to obtain NPCs and BMSCs. BMSCs were identified by flow cytometry, and its multipotent differentiation ability was proved by the induction of osteogenic, adipogenic and chondrogenic differentiation. NPC exosomes were extracted by differential centrifugation and identified by transmission electron microscope, and western blot analyses of exosomes surface makers, such as CD81, Tsg101. NPC exosomes and MSCs were labeled by fluorescent dye CM-Dil and CM-Dio respectively and incubated for 24h. Fluorescence microscopy was used to detect the uptake of NPC exosomes by MSCs. The third generation BMSCs were divided into three groups: exosomes group(treated with NPCs exosomes, 50μg/ml), coculture group(indirect cocultured with NPCs) and control group(no treatment). Quantitative RT-PCR(qRT-PCR) was performed to detect the expressions of Collagen Ⅱ, Aggrecan and SOX9 in exosomes group and control group in 7, 14, 21 days. QRT-PCR was performed to detect the expressions of Collagen Ⅱ, Aggrecan and SOX9 in three groups at 14d. Results: The third generation NPCs were polygonal. The third generation BMSCs were spindle and regular. BMSCs were positive for CD29(99.77%), CD44(93.97%), CD90(99.67%) and negative for CD34(0.82%), CD45(0.68%). BMSCs showed the ability to differentiate into osteoblasts, adipocytes and chondrocytes. The NPCs exosomes were revealed as round-shaped vesicles with double-layer membrane structure and <100nm in diameter under the transmission electron microscope. It was positive for CD81, Tsg101 and negative for calnexin. CM-DIL labeled NPCs exosomes could be internalized by CM-DIO labeled BMSCs under fluorescence microscopy. The mRNA expressions of Collagen Ⅱ, Aggrecan and SOX9 in exosomes group were higher than those in control group in 7, 14, 21 days(P<0.05). The mRNA expressions of Collagen Ⅱ, Aggrecan and SOX9 in coculture group were higher than those in control group, lower than those in exosomes group(P<0.05). Conclusions: Rat NPC exosomes can induce MSCs differentiate into NP-like cells in vitro, and its effects are better than indirect coculture system. It can provide an effective source of seed cells for intervertebral disc tissue engineering.