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

摘要/Abstract

摘要： 旨在建立西门塔尔牛表皮干细胞(epidermal stem cell，EpSCs)分离培养体系，探究其生物学特性，为西门塔尔牛种质资源保存提供新方法，为干细胞治疗研究提供种子细胞。采用3～4月龄西门塔尔牛背部表皮，通过酶消化法和组织贴壁法两种方法分离培养西门塔尔牛EpSCs，绘制EpSCs生长曲线探讨其增殖能力。通过免疫荧光鉴定EpSCs表面标记物(ITG β1、P63和CD71)，通过RT-PCR分析EpSCs特异性基因(ITG β1、KRT19和ITG α6)表达情况。通过体外诱导EpSCs分化为脂肪细胞、成骨细胞和软骨细胞检测其分化潜能。结果显示，组织块贴壁法获得的EpSCs纯度较低，酶消法获得的EpSCs纯度较高，细胞传代至P28代时开始逐渐老化。EpSCs细胞生长曲线呈“S”型。免疫荧光结果显示EpSCs表面特异性标记物ITG β1、P63和CD71呈阳性表达，RT-PCR结果显示EpSCs高度表达ITG β1、KRT19和ITG α6，不表达CD31基因。EpSCs诱导分化脂肪细胞时，产生可被油红O着色的脂滴，经鉴定细胞阳性表达脂肪细胞PPAR-γ和LPL特异性基因。EpSCs诱导分化成骨细胞时，形成可被茜素红染色呈橙红色的钙结节，且细胞阳性表达CollagenⅠ和RUNX2基因。EpSCs诱导分化软骨细胞时，透明状软骨基质经阿利新蓝染色呈蓝色，且细胞阳性表达软骨细胞特异性基因BGN和CollagenⅡ。上述结果表明，成功从西门塔尔牛胎牛表皮中分离得到生长状态良好的西门塔尔牛EpSCs，且具有向脂肪、成骨和软骨细胞等中胚层细胞分化的潜能，建立了西门塔尔牛EpSCs体外分离培养体系。

关键词: 西门塔尔牛, 表皮干细胞, 分离培养, 诱导分化

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

中图分类号:

S823.2

牛锐利, 宋哈楠, 吴月, 王育南, 高也凡, 关伟军, 宗宪春. 西门塔尔牛表皮干细胞的分离培养与生物学特性研究[J]. 畜牧兽医学报, 2023, 54(1): 168-177.

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.

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