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

摘要/Abstract

摘要： 【目的】研究北京油鸡角膜缘干细胞（LSCs）分离、培养、鉴定及多向分化潜能等生物学特性。【方法】取10胚龄健康北京油鸡鸡胚的角膜缘，采用分散酶Ⅱ（dispaseⅡ）联合胰蛋白酶二步法分离细胞，进行体外培养，绘制生长曲线，通过免疫荧光和RT-PCR技术鉴定LSCs特异性标志物细胞角蛋白3（CK3）、CK12、细胞周期调控蛋白p63、ATP结合盒转运蛋白（ABCG2）和细胞角蛋白19（CK19）的表达；通过成骨和成软骨诱导检测其多向分化潜能。【结果】获得的北京油鸡细胞折光性强、贴壁后呈多角形，生长曲线呈典型的S型，群体倍增时间（PDT）随着代次的升高逐渐下降；免疫荧光检测结果表明，分离的细胞中ABCG2、p63和CK19均呈阳性表达，CK3和CK12不表达；RT-PCR检测结果表明，分离的细胞表达p63、CK19、ABCG2，不表达CD45，说明分离的细胞为LSCs。LCSs成骨诱导分化后，可见被茜素红染色的矿化钙结节，RT-PCR检测结果表明其表达成骨关键基因骨桥蛋白（OPN）和Ⅰ型胶原蛋白（Col-Ⅰ）；成软骨诱导分化后，可见被阿利新蓝染色的软骨组织，RT-PCR检测结果表明其表达成软骨细胞关键基因转录因子性别决定区Y框9（SOX-9）和Ⅱ型胶原蛋白（Col-Ⅱ）。【结论】本研究成功从10胚龄北京油鸡胚胎角膜缘中分离LSCs，且其具有良好的增殖活力及成骨和成软骨能力，结果可为禽类LSCs的资源保存研究提供参考。

关键词: 北京油鸡; 角膜缘干细胞; 生物学特性; 诱导分化

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

中图分类号:

S858.292

高也凡, 宋哈楠, 王育南, 吴月, 牛锐利, 宗宪春, 关伟军. 北京油鸡角膜缘干细胞分离培养及分化潜能研究[J]. 中国畜牧兽医, 2022, 49(9): 3372-3381.

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 and Veterinary Medicine, 2022, 49(9): 3372-3381.

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