牙龈间充质干细胞来源外泌体对成骨样细胞MC3T3-E1体外增殖和成骨分化的影响

Effects of gingival mesenchymal stem cells derived exosomes on proliferation and osteogenic differentiation of osteoblast-like cell MC3T3-E1

背景牙龈间充质干细胞(gingival mesenchymal stem cells,GMSCs)来源外泌体具有与GMSCs相似的生物学功能,在组织工程领域具有良好的应用前景,但目前缺乏其在骨再生中应用的研究报道。目的 探讨GMSCs来源外泌体对成骨样细胞MC3T3-E1体外增殖和成骨分化的影响。方法 体外培养GMSCs,超速离心法提取GMSCs来源外泌体。共聚焦显微镜下观察MC3T3-E1细胞对外泌体的内化摄取。CCK-8法和划痕实验分别检测不同浓度外泌体作用下MC3T3-E1细胞增殖和迁移变化情况。外泌体作用于MC3T3-E1细胞后,碱性磷酸酶(alkaline phosphatase,ALP)染色检测ALP活性,茜素红染色及定量实验检测钙结节形成状况;RT-qPCR法检测成骨相关基因ALP、COL1A1、RUNX2的m RNA表达水平。结果 提取的GMSCs来源外泌体呈现类圆形膜状结构,大小在100 nm左右,表达CD63和Tsg101。外泌体能被MC3T3-E1摄取。GMSCs来源外泌体能够促进MC3T3-E1细胞的增殖和迁移(P<0.05),且呈剂量依赖性;外泌体作用下,MC3T3-E1细胞ALP活性增加(P<0.05),钙结节形成增多(P<0.05),成骨相关基因ALP、COL1A1、RUNX2的mRNA表达水平明显上调(P<0.05)。结论 GMSCs来源外泌体具有增强MC3T3-E1增殖及成骨分化的作用,这为GMSCs来源外泌体应用于骨再生治疗奠定基础。

Background Exosomes derived from gingival mesenchymal stem cells(GMSCs) have similar biological functions to GMSCs, which is a promising treatment for tissue repair. However, the research of GMSCs derived exosomes on bone regeneration is rarely reported. Objective To investigate the effects of GMSCs-derived exosomes on the proliferation and osteogenic differentiation of osteoblast-like cells MC3T3-E1 in vitro. Methods GMSCs were cultured, and exosomes were extracted by ultracentrifugation from supernatants. Uptake of exosomes by MC3T3-E1 was observed under an inverted fluorescence microscope.CCK-8 assay and scratch test were used to detect the changes of proliferation and migration of MC3T3-E1 with different concentrations of exosomes. After exosomes acted on MC3T3-E1, the activity of alkaline phosphatase(ALP) was detected by ALP staining, and the formation of calcium nodules was detected by alizarin red staining and quantitative experiments. RT-qPCR method was used to detect the mRNA expression levels of osteogenesis related genes ALP, COL1A1(Collagen Type I Alpha 1) and RUNX2(RUNX Family Transcription Factor 2). Results The extracted exosomes derived from GMSCs presented a round membranous structure with a size of about 100 nm and expressed CD63 and Tsg101 marker proteins. GMSCs derived exosomes could promote the proliferation and migration of MC3T3-E1(P<0.05) in a dose-dependent manner. The ALP activity of MC3T3-E1 and the calcium nodule Nodal formation increased under the action of exosomes, and the mRNA expression levels of osteogenic related genes ALP, COL1A1 and RUNX2 were significantly up-regulated(all P<0.05). Conclusion GMSCs derived exosomes can promote the proliferation and osteogenic differentiation of MC3T3-E1, which lays the foundation for the application of GMSCs derived exosomes to bone regeneration treatment.