西门塔尔牛表皮干细胞的分离培养与生物学特性研究

doi:10.11843/j.issn.0366-6964.2023.01.016

Abstract

Abstract: The study aimed to establish a system for isolation and culture of epidermal stem cells (EpSCs) of Simmental cattle, study its biological characteristics, explore a new method for the conservation of Simmental germplasm resources, and provide seed cells for stem cell therapy research. The EpSCs were isolated from the dorsal epidermis of 3-to 4-month-old Simmental cattle by enzyme digestion method and tissue adhesion method. Simmental cattle EpSCs were then cultured to draw the growth curve and to further investigate the proliferation ability. EpSCs surface markers (ITG β1, P63 and CD71) were identified by immunofluorescence, and the expression analysis of specific genes (ITG β1, KRT19 and ITG α6) were performed by RT-PCR. Simmental cattle EpSCs were induced to differentiate into adipocytes, osteoblasts and chondrocytes in vitro for detecting their differentiation potential. The results showed that the purity of EpSCs obtained by tissue adhesion method was much lower than that obtained by enzyme digestion method, and they began to age gradually when passed to P28 generation. The growth curve of EpSCs was shaped as “S”. Immunofluorescence result showed that positive expression of surface specific markers in EpSCs such as ITG β1, P63 and CD71, RT-PCR showed that EpSCs highly expressed gene ITG β1, KRT19 and ITG α6 but CD31. When induced to adipocytes, EpSCs produced lipid droplets that could be stained by Oil red O and positively expressed specific genes of adipocyte PPAR-γ and LPL. When induced to osteoblasts, EpSCs produced calcium nodules that could be stained as orange red by Alizarin red, and positively expressedgenes such as Collagen Ⅰ and RUNX2. When induced to chondrocytes, they produced hyaline cartilage matrix that could be stained as blue by Alcian blue and positively expressed specific genes BGN and Collagen Ⅱ of chondrocytes. In this research, Simmental cattle EpSCs in good condition were successfully isolated from epidermis of fetal bovine with the potential to differentiate into osteogenesis, cartilage and adipose cells, the isolation and culture system in vitro for Simmental EpSCs was established.

Key words: Simmental cattle, epidermal stem cells, isolation and culture, induction differentiation

CLC Number:

S823.2

NIU Ruili, SONG Hanan, WU Yue, WANG Yunan, GAO Yefan, GUAN Weijun, ZONG Xianchun. Isolation, Culture and Biological Characteristics of Epidermal Stem Cells from Simmental Cattle[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(1): 168-177.

References 31

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Isolation, Culture and Biological Characteristics of Epidermal Stem Cells from Simmental Cattle

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