西门塔尔牛牙龈间充质干细胞的分离鉴定及诱导分化

本研究旨在建立西门塔尔牛牙龈间充质干细胞（gingival mesenchymal stem cells,GMSCs）体外分离培养体系,并对其生物学特性进行探讨,为细胞治疗、再生医学等提供种子细胞。取14周龄西门塔尔牛牙龈,采用0.1%Ⅳ型胶原酶消化法分离GMSCs,进行体外培养和冷冻保存,测定冻存前后细胞活率,MTT检测法绘制GMSCs生长曲线;通过免疫荧光、流式细胞术和PCR等方法鉴定西门塔尔牛GMSCs特异性标记物（CD29、CD44、CD73、CD90、CD105、CD166和STRO-1）,并通过体外成脂、成骨诱导检测其多向分化潜能。结果表明,西门塔尔牛GMSCs折光性强、贴壁后呈长纺锤形;冻存前和复苏后细胞的活率检测结果显示,细胞复苏后的活率均小于冻存前的活率,但均维持在85%以上,细胞生长曲线呈典型的S型;免疫荧光结果显示,西门塔尔牛GMSCs表达CD29、CD105和STRO-1,不表达CD31;PCR结果显示,西门塔尔牛GMSCs表达CD44、CD73、CD90和CD166,不表达CD34和CD45;流式细胞术结果显示,西门塔尔牛GMSCs高表达CD73（99.94%）、CD90（99.87%）和CD105（99.90%）,几乎不表达CD34（1.16%）和CD45（1.18%）;成脂诱导分化后,可见大小不一的脂滴,脂滴可被油红O染色,且PCR检测结果表明其表达成脂关键基因过氧化物酶体增殖因子活化受体-γ（PPAR-γ）和脂蛋白酶（LPL）;成骨诱导分化后,可见被茜素红染色的矿化结节,且PCR检测结果表明其表达成骨关键基因Ⅰ型胶原蛋白（CollagenⅠ）和骨桥蛋白基因（OPN）。综上,本研究成功分离出西门塔尔牛GMSCs,建立了西门塔尔牛GMSCs体外分离、培养体系,并通过成脂和成骨诱导分化证明其具有多向分化潜能,结果可为再生医学和牙齿修复等研究提供参考。

Isolation,Identification and Induction Differentiation of Gingival Mesenchymal Stem Cells in Simmental Cattle

The purpose of this study was to establish the gingival mesenchymal stem cells (GMSCs) isolation and culture system in vitro,and to explore their biological characteristics,so as to provide seed cells for cell therapy and regenerative medicine.14 weeks of Simmental cattle gum was used to separate GMSCs digested by 0.1% type Ⅳ collagenase digestion,and then in vitro culture and cryopreservation were performed,cell viability was measured before and after cryopreservation,GMSCs growth curve was plotted by MTT assay.GMSCs specific markers (CD29,CD44,CD73,CD90,CD105,CD166 and STRO-1) were identified by immunofluorescence,flow cytometry and PCR,and their multidirectional differentiation potential was detected by adipogenic and osteogenic induction in vitro.The results showed that GMSCs of Simmental cattle had strong refraction and long spindle shape after adherent to the wall.The results of cell viability test before and after cryopreservation showed that the cell viability after cryopreservation was lower than that before cryopreservation,but remained above 85%,and the cell growth curve showed a typical S type.Immunofluorescence results showed that GMSCs of Simmental cattle expressed CD29,CD105 and STRO-1,but not CD31.PCR results showed that GMSCs of Simmental cattle expressed CD44,CD73,CD90 and CD166,but not CD34 and CD45.Flow cytometry results showed that CD73 (99.94%),CD90 (99.87%) and CD105 (99.90%) were highly expressed by Simental bovine GMSCs,while CD34 (1.16%) and CD45 (1.18%) were almost not expressed.After adipogenic differentiation,lipid droplets of different sizes were observed,which could be stained with oil red O,and the PCR results showed that they expressed the key lipid genes peroxidase proliferation factor activated receptor-γ (PPAR-γ) and lipoprotein enzyme (LPL).After the osteogenetic differentiation,mineralized nodules were displayed by Alizarin red staining,and PCR detection results showed that osteogenesis key genotype Collagen Ⅰ and osteopontin (OPN) gene were expressed.In conclusion,the GMSCs of Simmental cattle were successfully isolated in this study,and the isolation and culture system of GMSCs of Simmental was established in vitro.Moreover,the multidirectional differentiation potential was proved through adipogenic and osteogenic induction and differentiation,the results could provide references for studies on regenerative medicine and tooth repair.