猫角膜缘干细胞不同采集、分离与培养鉴定方法的对比研究

doi:10.11843/j.issn.0366-6964.2023.07.039

畜牧兽医学报 ›› 2023, Vol. 54 ›› Issue (7): 3091-3101.doi: 10.11843/j.issn.0366-6964.2023.07.039

猫角膜缘干细胞不同采集、分离与培养鉴定方法的对比研究

胥辉豪^1,2,3, 冯雪倩¹, 朴雪玲¹, 慎晓军¹, 郑小波¹, 杨恒¹, 林珈好^3*, 金艺鹏^3*, 林德贵^3*

1. 西南大学动物医学院, 重庆 402460;
2. 西南大学发光分析与分子传感教育部重点实验室, 重庆 400700;
3. 中国农业大学动物医学院, 北京 100193

收稿日期:2022-12-10 出版日期:2023-07-23 发布日期:2023-07-21
通讯作者: 林德贵,主要从事兽医外科学研究,E-mail:csama@sina.com;金艺鹏,主要从事兽医眼科学、牙科学、野生动物疾病学研究,E-mail:yipengjin@vip.sina.com;林珈好,主要从事中兽医学研究,E-mail:jiahao_lin@cau.edu.cn
作者简介:胥辉豪(1983-),男,重庆人,高级实验师,博士,主要从事兽医外、产科学、兽医眼科学与肿瘤学研究,E-mail:xuhuihao2dai@163.com;Tel:023-68251603
基金资助:
国家自然科学基金（31972730）；西南大学实验技术研究项目（SYJ2020013）；深圳市瑞鹏公益基金会高校青年教师（小动物临床）科研基金（RPJJ2020001）

Comparative Study on Different Methods of Collection, Isolation, Culture and Identification of Feline Limbal Stem Cells

XU Huihao^1,2,3, FENG Xueqian¹, PIAO Xueling¹, SHEN Xiaojun¹, ZHENG Xiaobo¹, YANG Heng¹, LIN Jiahao^3*, JIN Yipeng^3*, LIN Degui^3*

1. College of Veterinary Medicine, Southwest University, Chongqing 402460, China;
2. Key Laboratory of Luminescence Analysis and Molecular Sensing (Southwest University), Ministry of Education, Chongqing 400700, China;
3. College of Veterinary Medicine, China Agricultural University, Beijing 100193, China

Received:2022-12-10 Online:2023-07-23 Published:2023-07-21

摘要/Abstract

摘要： 本研究旨在对比不同的分离与培养方法体外培养猫角膜缘干细胞（feline limbal stem cells，FLSCs）的效果，建立使FLSCs持续稳定增殖、结构及功能正常的最佳分离与培养体系。在手术显微镜下活体采集猫角膜缘组织，分别使用组织贴壁法（T法）、混合酶消化法（N法）、混合酶消化法联合组织贴壁法（NT法）分离FLSCs，鉴定LSCs标志蛋白ABCG2，以筛选最适分离方法。用BC培养基、DLM培养基和SCM培养基进行细胞培养，连续7 d细胞计数、观察细胞形态，选取LSCs的阳性标志物p63、波形蛋白和上皮分化标记物CK3、CK12定性分析第3、4、5代培养细胞。结果显示：3种方法分离的细胞中，ABCG2均呈阳性表达，NT法表达最强，表明3种方法均可分离FLSCs，NT法消化分离效果最好。3种培养基均可培养FLSCs，细胞形态无差异，细胞在DLM组中增殖速度最快，更快到达细胞生长峰值，与其他两组比，差异显著（P<0.05）。FLSCs生长曲线图呈典型"S"型，前72 h为迟缓期，72~216 h为对数生长期，216~264 h则基本处于平稳期，从264 h开始进入衰亡期。FLSCs群体倍增时间为38.9 h，平均最大增殖浓度为5.66×10⁵ cells·mL^-1。第3、4和5代FLSCs中波形蛋白、p63均呈强阳性表达，CK3和CK12不表达。使用NT法分离FLSCs，并在DLM中进行培养，可建立稳定增殖的FLSCs体外培养体系。

关键词: 猫, 角膜缘干细胞, 分离方法, 培养体系

Abstract: The purpose of this study was to compare the effects of different isolation and culture methods on the culture of feline limbal stem cells (FLSCs) in vitro, and to establish the best isolation and culture system for the continuous and stable proliferation, normal structure and function of FLSCs. The limbal tissues of cats were collected under surgical microscope. Three different digestion methods which were the tissue adhesion method (T method), the mixed enzyme digestion method (N method) and the mixed enzyme digestion methods combined with tissue adhesion method (NT method) were used to separate FLSCs, to identify the expression of LSCs marker protein ABCG2, in order to screen the most suitable method for separation. Cells were cultured through three kinds of complete media which were BC, DLM, and SCM, the cell count and the cell morphology was observed every day for 7 days, and the positive markers of LSCs p63, vimentin and epithelial differentiation markers CK3, CK12 were selected to qualitatively analyze the 3rd, 4th, and 5th generation cells. The results showed that the expression of ABCG2 from cells separated by three methods were positive and that of NT method expressed the most positive, indicating that three methods can all be used to separate FLSCs, and the separation effect of NT method was the best. FLSCs can be cultured in all three media, with no difference in cell morphology. Cells proliferation speed were the fastest in group DLM. Compared with the other two groups, time of cell reaching the peak cell growth was shorter in DLM, and the difference was significant (P<0.05). The growth curve of FLSCs was showed a typical "S" type. The first 72 h of LSCs growth was in the lag phase, the 72-216 h was in the logarithmic growth phase, the 216-264 h was basically in the plateau phase, and the cells entered the decay phase at the 264th hour. The population doubling time of FLSCs was 38.9 h, and the average maximum proliferation concentration was 5.66×10⁵cells·mL^-1. The 3rd, 4th and 5th generation FLSCs showed strong positive expression of vimentin and p63, but CK3 and CK12 didn't express. FLSCs can be isolated by NT method and cultured in DLM to establish a stable proliferation system of FLSCs in vitro.

Key words: feline, limbal stem cells, isolation method, culture system

中图分类号:

S857.6

胥辉豪, 冯雪倩, 朴雪玲, 慎晓军, 郑小波, 杨恒, 林珈好, 金艺鹏, 林德贵. 猫角膜缘干细胞不同采集、分离与培养鉴定方法的对比研究[J]. 畜牧兽医学报, 2023, 54(7): 3091-3101.

XU Huihao, FENG Xueqian, PIAO Xueling, SHEN Xiaojun, ZHENG Xiaobo, YANG Heng, LIN Jiahao, JIN Yipeng, LIN Degui. Comparative Study on Different Methods of Collection, Isolation, Culture and Identification of Feline Limbal Stem Cells[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(7): 3091-3101.

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猫角膜缘干细胞不同采集、分离与培养鉴定方法的对比研究

Comparative Study on Different Methods of Collection, Isolation, Culture and Identification of Feline Limbal Stem Cells

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