人羊膜和牙周膜来源间充质干细胞免疫表型与干性标志基因表达的比较分析  

Comparative analysis of the immunophenotypes and expression of stemness marker genes in human mesenchymal stem cells derived from human amniotic membrane and periodontal ligament

作　　者：陈辉 杨琨 蒋珊珊 林风琴 何青 方宁 余丽梅 CHEN Hui;YANG Kun;JIANG Shanshan;LIN Fengqin;HE Qing;FANG Ning;YU Limei(Key Laboratory of Cell Engineering in Guizhou Province/the Guizhou Province Foundation and Clinical Research Technology Innovation Team on Amniotic and Bone Marrow Mesenchymal Stem Cell,Affiliated Hospital of Zunyi Medical University,Zunyi,Guizhou 563003,China;Guizhou Biotherapy Talent Base,Zunyi Medical University,Zunyi,Guizhou 563003,China;Department of Periodontology,Hospital of Stomatology,Zunyi Medical University,Zunyi,Guizhou 563003,China)

机构地区：[1]遵义医科大学附属医院贵州省细胞工程重点实验室/贵州省羊膜与骨髓间充质干细胞基础与临床研究科技创新团队,贵州遵义563003 [2]遵义医科大学贵州省生物治疗人才基地,贵州遵义563003 [3]遵义医科大学附属口腔医院牙周科,贵州遵义563003

出　　处：《重庆医学》2020年第14期2249-2254,共6页Chongqing medicine

基　　金：国家自然科学基金项目(81260507、81760199);贵州省科技厅科技重大专项{[黔科合重大专项]字(2011)6002}。

摘　　要：目的研究羊膜和牙周膜来源间充质干细胞的免疫表型及干性基因表达的异同。方法取羊膜和牙周膜各3例,采用酶消化法分离细胞并进行细胞传代培养,获取第3代人羊膜间充质干细胞(hAMSCs)与人牙周膜干细胞(hPDLSCs),流式细胞术检测分子免疫表型,实时定量PCR检测干性标志基因表达水平。结果原代培养的hAMSCs生长速度快于hPDLSCs,后者形态均一;第2、3代细胞均生长良好,每3天可传代1次,第3代hAMSCs和hPDLSCs都为均一的长梭形,呈旋涡状排列,均高表达CD90、CD73、CD105和CD44,表达百分率高于98%;hAMSCs的CD34、CD19、CD45、CD11b、人类白细胞抗原DR(HLA-DR)总阳性表达率低于hPDLSCs(P<0.05)。两种干细胞均不表达/低表达Sox2 mRNA,而Nanog、Oct4和C-myc mRNA呈中高水平表达,差异无统计学意义(P>0.05)。结论hAMSCs和hPDLSC具有相似的形态特征和免疫表型,且干性基因表达无明显差异,hAMSCs的免疫原性可能更低,应用前景较大。Objective To explore the similarities and differences of immunophenotypes and expression of stemness marker genes in mesenchymal stem cells derived from amniotic membrane and periodontal ligament.Methods Three samples of amniotic membrane and periodontal ligament were taken.The third generation of human amniotic mesenchymal stem cells(hAMSCs)and human periodontal ligament stem cells(hPDLSCs)were obtained by pancreatin digestion and subculture.The flow cytometry was used to detect cellular molecular immunophenotype,and the real-time quantitative PCR was used to detect the expression level of stem cell marker genes.Results The growth rate of primary cultured hAMSCs was faster than that of hPDLSCs,and the latter was homogeneous in morphology.The second and third generations cells all grew well,and could be subcultured once every three days.The third generation hAMSCs and hPDLSCs were both uniformly long spindle-shaped cells that arranged in a spiral pattern.The positive expression percentages of CD90,CD73,CD105 and CD44 were all higher than 98%in the third generation hAMSCs and hPDLSCs.The total positive expression rate of CD34^+CD19^+CD45^+CD11b^+HLA-DR^+of hAMSCs was lower than that of hPDLSCs,and the difference was statistically significant(P<0.05).The hAMSCs and hPDLSCs did not express or express low level of Sox2,while Nanog,Oct4 and C-myc mRNA were expressed at medium to high levels,and there was no significant difference between them(P<0.05).Conclusion hAMSCs and hPDLSC have similar morphological features and immunophenotypes,and there is no significant difference in expression of stem cell markers.Compared to hPDLSC,hAMSCs have lower immunogenicity,which will be widely used.

关 键 词：间充质基质细胞 羊膜 牙周组织 免疫表型分型 干性基因 

分 类 号：R319[医药卫生—基础医学]