北京油鸡角膜缘干细胞分离培养及分化潜能研究

doi:10.16431/j.cnki.1671-7236.2022.09.011

Abstract

Abstract: 【Objective】 The aim of this study was to explore the biological characteristics of limbal stem cells (LSCs) isolation, culture, identification and multidirectional differentiation potential of Beijing You chickens.【Method】 Limbus cornea of 10-day-old healthy Beijing You chicken embryos was isolated by dispase Ⅱ combined with trypsin two-step method.The cells were cultured in vitro and the growth curve was drawn.The expression of LSCs specific markers cytokeratin 3 (CK3), CK12, cell cycle regulatory protein p63, ATP binding box transporter (ABCG2) and cytokeratin 19 (CK19) were identified by immunofluorescence and RT-PCR.Multidirectional differentiation potential was detected by osteogenic and chondrogenic induction.【Result】 The cells of Beijing You chickens showed strong refraction, polygonal shape, typical S shaped growth curve, and population doubling time (PDT) was decreased with the increase of generations.Immunofluorescence assay showed that ABCG2, p63 and CK19 were positive, while CK3 and CK12 were negative.The results of RT-PCR showed that the isolated cells expressed p63, CK19 and ABCG2, but did not express CD45, indicating that the isolated cells were LSCs.After osteogenic induction and differentiation of LCSs, mineralized calcium nodules stained with alizarin red could be seen.RT-PCR results showed that osteopontin (OPN) and collagen type Ⅰ(Col-Ⅰ) which were key osteogenic genes were expressed in LCSs.After induction and differentiation of chondroblasts, the chondroblasts were stained with alicin blue.The results of RT-PCR showed that the expression of key chondroblast gene transcription factors Y-frame 9 (SOX-9) and type Ⅱ collagen (Col-Ⅱ) were detected.【Conclusion】 In this study, LSCs were successfully isolated from limbus cornea of 10-day-old Beijing You chicken embryos, and they had good proliferative activity and osteogenic and chondrogenic abilities.The results could provide references for the conservation of poultry LSCs resources.

Key words: Beijing You chickens; limbal stem cells; biological characteristics; induced differentiation

CLC Number:

S858.292

GAO Yefan, SONG Hanan, WANG Yunan, WU Yue, NIU Ruili, ZONG Xianchun, GUAN Weijun. Study on the Isolation, Culture and Differentiation Potential of Limbal Stem Cells from Beijing You Chickens[J]. China Animal Husbandry & Veterinary Medicine, 2022, 49(9): 3372-3381.

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Study on the Isolation, Culture and Differentiation Potential of Limbal Stem Cells from Beijing You Chickens

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