西门塔尔牛牙龈间充质干细胞的分离鉴定及诱导分化

doi:10.16431/j.cnki.1671-7236.2021.03.009

Abstract

Abstract: 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.

Key words: Simmental cattle; gingival mesenchymal stem cells; isolation and culture; induction differentiation

CLC Number:

Q254

SUN Yuchen, XU Long, YIN Jiahui. Isolation,Identification and Induction Differentiation of Gingival Mesenchymal Stem Cells in Simmental Cattle[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(3): 855-864.

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

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