猪耳廓软骨板细胞的体外传代培养  

In vitro Subculture of Cartilage cells of Pig(Sus scrofa) auricle

作　　者：伍仲平 张震[1] 张慧[1] 段艳宇[1] 

机构地区：[1]江西农业大学猪遗传改良与养殖技术国家重点实验室,南昌330044

出　　处：《农业生物技术学报》2016年第6期799-805,共7页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(No.31301950);江西省研究生创新专项资金项目(No.YC2014-S191)

摘　　要：耳廓是哺乳动物听觉器官的重要组成部分。猪（Sus scrofa）耳廓大小经历长期的人工驯养和选育后变异很大。目前,对猪耳廓大小的研究主要集中在遗传机理的解析,对其在细胞水平的研究还尚未完善。本研究通过酶法分离得到猪耳廓软骨板细胞并在体外培养,将原代猪耳廓细胞连续培养,用流式细胞仪分别检测从3-10代耳廓软骨板细胞的大小和分布情况,以及耳廓软骨板细胞与软骨干细胞阳性标记物CD44和CD90的结合能力。同时,将3-10代细胞分别与Annexin V-FITC/PI（细胞凋亡试剂盒）孵育,用流式细胞仪进行凋亡特性分析。通过流式细胞检测发现,体外培养的软骨板细胞由软骨细胞（P1）和软骨前体细胞（P2）组成。P1细胞占大多数（69.4%）,呈纤维状;P2细胞为少数（11.0%）,呈椭圆状。与CD44和CD90结合分析表明,P1细胞几乎不能与CD44结合,绝大部分P2细胞能与CD44结合,P1和P2细胞均能与CD90相结合,P2细胞结合能力略强于P1细胞。分别对两类细胞进行细胞凋亡分析后发现,P1细胞体外培养只有少量细胞凋亡或坏死,活细胞率达（96.61±1.32）%,说明P1细胞在体外传代过程中能维持稳定状态;P2细胞处于晚期凋亡和坏死细胞的比例〉83%,说明P2细胞在体外培养条件下细胞状态不稳定。本研究结果表明,体外建立一套针对于软骨前体细胞培养的体系很有必要,以保证软骨前体细胞可以持续增殖、并可向软骨细胞转化。本研究为今后以猪耳廓作为模型开展人类弹性软骨再生医学研究提供了基础依据。For mammals, ear is an important auditory organ for the recognition of sound waves and the identification of orientation. Considering that ear size of pig （Sus scrofa） has a very large variation after long-term domestication and artificial breeding, it is an ideal model animal for human auricular disease. So far, the study of pig ear size has mainly focused on the genetic mechanism, yet the research on the pig auricle cells is limited. In this study, using enzymatic method, cells in cartilage of pig ear was isolated and cultured in vitro. The cells in cartilage of pig ear were continuously subcultured and detected their size and distribution during the 3-10 passage by flow cytometry, respectively. The cells in cartilage of pig ear were mainly devided into fibrillar type （P1） and oval type （P2）. P1 was of bigger size account for the vast majority of the cells （69.4%）, and P2 was the minority （11.0%）. Then the combination capacity of the 2 kinds of cells was detected using the positive markers of cartilage stem cells CD44 and CD90. The results showed that cells P1 could hardly combine with CD44, cells P2 had a strong combination capacity with CD44. However, both P1 and P2 could bind with CD90, which the binding capacity of P2 was slightly stronger than P1. According to the analysis results, pig auricle cells were comprised by chondrocytes （P1） and cartilage progenitor cells （P2）. Subsequently, in order to study the quantity and physiological state changes of P1 and P2 cells during passages, the cells were incubated with FITC-Annexin V/PI to analyze apoptosis using flow cytometry. The result showed that apoptosis was a common phenomenon in the pig ear cartilage cells cutured in vitro from the 3-6 passages with a rising trend of apoptosis, and after the 6 generations, the situation of apoptosis was basically constant. Further apoptosis analysis result showed that P1 cells were only a small amount of apoptosis or necrosis, with cell viability of （96.61±1.32）%, indicating that P1 cell c

关 键 词：猪耳廓软骨板细胞 流式细胞术 细胞凋亡 

分 类 号：Q255[生物学—细胞生物学]