骨髓间充质干细胞来源的外泌体促进牙周再生的体外研究

Study of periodontal regeneration promoted by exosome from BMMSC in vitro

目的研究人骨髓间充质干细胞(human bone marrow mesenchymal stem cells,hBMMSCs)分泌的外泌体(exosome)对人牙周膜干细胞(human periodontal ligament stem cells,hPDLSCs)增殖和分化功能的影响,为进一步明确hBMMSCs促进牙周组织再生的机制提供实验基础和证据。方法分别用酶消化法和离心法培养hPDLSCs和hBMMSCs,并用流式细胞术检测其间充质表面标志物。收集培养的第3代hBMMSCs上清液,利用试剂盒提取exosome,电镜下观察其结构,Western Bolt法检测其CD63表达水平。用hBMMSCs分泌的exosome处理hPDLSCs设为实验组,四甲基偶氮唑盐(MTT)法检测hPDLSCs的增殖情况,克隆形成率检测多克隆形成能力,成骨诱导后碱性磷酸酶(ALP)染色和茜素红染色并定量,同时实时荧光定量PCR(qPCR)检测成骨相关基因表达。结果流式细胞术检测,hPDLSCs阳性表达CD29、CD44、CD90、CD146和Stro-1,hBMMSCs阳性表达CD29、CD44、CD90和CD106,两者均阴性表达CD14、CD34和CD45。hBMMSCs分泌的exosome电镜下呈小球状,Western Bolt法检测其强表达CD63。实验组hPDLSCs经MTT法检测其增殖速率和克隆形成能力显著高于对照组(P<0.05)。实验组hPDLSCs成骨诱导后ALP染色、茜素红染色定量表达显著强于对照组(P<0.05)。qPCR检测成骨基因Runx2、OCN、ALP、BSP和Col-Ⅰ的表达,实验组显著高于对照组(P<0.05)。结论 hBMMSCs分泌的exosome可促进牙周膜干细胞的体外增殖和成骨分化能力。

Objective To study the influence of exosome secreted by the human bone marrow mesenchymal stem cells （hBMMSCs） on proliferation and differentiation of periodontal ligament stem cells （hPDLSCs） , and to provide experi- mental basis and evidence of the mechanism of hBMMSCs in promoting periodontal tissue regeneration. Methods hP- DLSCs were cultured by enzyme digestion method and hBMMSCs were cultured by centrifugation method, and the immu- nophenotype was identified by flow cytometry. Supernatant of hBMMSCs in P3 was collected, cxosome was extracted by kit and observed by SEM, and CD63 level was tested by WB. The experiment group was treated by exosome of hBMMSCs and control group was treated by DD water, hPDLSCs proliferation was tested by MTT and colony unite forming was test-ed in 7 days. After osteogenetic induction, ALP and Aliz- arin Red staining were performed and quantitative analy- sis was carried. Meanwhile osteogenesis-related gene ex- pression was tested by qPCR. Results hPDLSCs posi- tively expressed CD29, CD44, CD90, CD146 and Stro-1; hBMMSCs positively expressed CD29, CD44, CD90 and CD106; both negatively expressed CD14, CD34 and CD45. Exosome secreted by hBMMSCs weresmall ball-shaped under SEM and expressed CD63 strongly by WB. The proliferation and colony unite forming of experi- mental group was significantly higher than control group （P 〈 0.05 ）. The quantitative expression of ALP and alizarin red staining after osteogenetic induction were significantly stronger than the control group （P 〈 0.05）. The osteogenetic gene expression, including Runx2, OCN, ALP, BSP and Col - I , was significantly higher than that of control group by qP- CR （P 〈 0.05）. Conclusion The exosome secreted by hBMMSCs can promote proliferation and osteogenesis of hP- DLSCs in vitro.