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郭晓楠,李云霞,郝晓丽,王茂俊,乌云毕力格,吴图雅,格根卓拉,张健,宋永利,赵高平,曹贵方,包斯琴,李喜和.2022.西伯利亚狍成纤维细胞建系及其生物学特性分析.动物学杂志,57(5):653-667.
西伯利亚狍成纤维细胞建系及其生物学特性分析
In Vitro Culture of Fibroblasts and Analysis of Their Biological Characteristics in Siberian Roe Deer
投稿时间:2021-11-19  修订日期:2022-09-09
DOI:10.13859/j.cjz.202205002
中文关键词:  西伯利亚狍  成纤维细胞  冷冻前及复苏后存活率  生长曲线  核型和G带
英文关键词:Siberian Roe Deer, Capreolus pygargus  Fibroblast  Survival rates before freezing and after resuscitation  Growth curve  Karyotype and G-band
基金项目:内蒙古自治区科技成果转化引导项目(No. 2020CG0078),奶山羊、奶绵羊良种扩繁技术产业化应用项目(No. 2019-自-戊=10)
作者单位E-mail
郭晓楠 内蒙古大学生命科学学院蒙古高原动物遗传资源研究中心 呼和浩特 010020 1009426569@qq.com 
李云霞 内蒙古赛科星家畜种业与 繁育生物技术研究院 呼和浩特 011517 liyunxia831130@163.com 
郝晓丽 内蒙古赛科星家畜种业与 繁育生物技术研究院 呼和浩特 011517 haoxiaoli_imau@163.com 
王茂俊 内蒙古大学生命科学学院蒙古高原动物遗传资源研究中心 呼和浩特 010020 2236651970@qq.com 
乌云毕力格 内蒙古大学生命科学学院蒙古高原动物遗传资源研究中心 呼和浩特 010020 1938303361@qq.com 
吴图雅 内蒙古大学生命科学学院蒙古高原动物遗传资源研究中心 呼和浩特 010020 1598463516@qq.com 
格根卓拉 内蒙古赛科星家畜种业与 繁育生物技术研究院 呼和浩特 011517 ggzora@163.com 
张健 河套学院 巴彦淖尔 015000 909719423@qq.com 
宋永利 内蒙古大学生命科学学院蒙古高原动物遗传资源研究中心 呼和浩特 010020 songyongli625@163.com 
赵高平 内蒙古赛科星家畜种业与 繁育生物技术研究院 呼和浩特 011517 gaopingzhao@126.com 
曹贵方 内蒙古赛科星家畜种业与 繁育生物技术研究院 呼和浩特 011517内蒙古农业大学兽医学院 呼和浩特 010018 guifangcao@126.com 
包斯琴 ①内蒙古大学 蒙古高原动物遗传资源研究中心 baosq@life.imu.edu.cn 
李喜和* 内蒙古大学生命科学学院蒙古高原动物遗传资源研究中心 呼和浩特 010020内蒙古赛科星家畜种业与 繁育生物技术研究院 呼和浩特 011517 lixh@imu.edu.cn 
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中文摘要:
      本研究以内蒙古大青山获得野生雄性和雌性西伯利亚狍(Capreolus pygargus)为实验材料,利用组织块贴壁培养法进行气管、肺和耳3种组织成纤维细胞原代建系,研究不同组织来源的细胞贴壁率、冷冻前及复苏后存活率、生长曲线,进一步绘制狍成纤维细胞核型图并分析其G带特征。实验结果显示,气管、肺和耳3种组织成纤维细胞增殖经历潜伏期、对数生长期、平台期三个阶段,细胞形态为梭形、三角形或不规则形,是典型成纤维细胞形态;成纤维细胞呈漩涡状生长,其中气管、耳成纤维细胞生长增殖能力最强、肺成纤维细胞增殖能力较弱,气管和耳组织来源成纤维细胞呈典型“S”型细胞生长特征。染色体核型及G带分析结果显示,雄性狍成纤维细胞染色体条数为2n = 70,其中,有34对常染色体,形态类型为12条近端着丝粒染色体(st),22条亚中着丝粒染色体(sm),1对性染色体,X染色体为中着丝粒染色体(m),Y染色体为近端着丝粒染色体(st),5条超数染色体(B);雌性狍成纤维细胞染色体条数为2n = 70,其中,有34对常染色体,其形态类型为29条亚中着丝粒染色体(sm),5条近端着丝粒染色体(st),1对为性染色体,X染色体为亚中着丝粒染色体(sm),8条超数染色体(B)。本研究成功建立了雄性和雌性西伯利亚狍气管、肺和耳3种组织来源的成纤维细胞系,在体外培养时生长状态良好且维持了细胞的遗传信息稳定性,绘制了西伯利亚狍雄性和雌性染色体核型及G带图谱,为将来更深入开展相关研究提供材料与基本技术支撑。
英文摘要:
      [Objective] Siberian Roe Deer (Capreolus pygargus) is a kind of important wildanimal with edible value, meidicinal and ornamental value for scientific research. To understand the biological characteristics of Siberian Roe Deer fibroblasts, trachea, lung and ear tissues were collected for cell culture. [Methods] In this study, wild male and female Siberian Roe Deers were collected as experimental materials from Daqingshan, Inner Mongolia Autonomous Region, China. The primary fibroblasts of trachea, lung and ear were established by tissue block monolayer culture method. The cell adherence rate, survival rate before freezing and after resuscitation, growth curve of cells from different tissue sources were explored, and the karyotype and the G-band characteristics were analyzed. Unless otherwise noted, all analyses were performed in at least triplicate. Student’s t-test was uses to calculate difference using Graphpad (Version 5.0) [Results] The experimental results showed that spindle-shaped or triangular cells were newly grown along the tissue blocks in the trachea and ear tissues of male roe deer after 2﹣4 days of culture. After 8﹣10 days of culture, the convergence degree of cells in the flask reached 60%, and after 11﹣12 days of culture, the convergence degree of cells in the flask reached 80% (Fig. 1). The morphology and growth of fibroblasts from trachea, ear and lung of the femalewere basically the same as those of the male (Fig. 2). The most densely populated fibroblasts from the trachea, ears and lungs were observed at P20, P12 and P9, respectively (Fig. 3). The adherence rates of fibroblasts from the three tissues increased gradually with time, and grew rapidly within 6 - 12 h. After 24 h, the adherence rates of fibroblasts from the trachea and ears were over 95%. The adherence rate of P3﹣P9 lung fibroblasts varied greatly, and the adherence rate reached more than 90% when the culture time was 24 h (Table 1). There was almost no difference in the cell survival rate of airway fibroblasts in different generations before cryopreservation, and the survival rates were all above 85%. There was little difference in cell survival rate between ear and lung fibroblasts in different generations before cryopreservation, and the survival rate was above 90%. The recovery rate of fibroblasts after freezing gradually decreased with the increase of cell passage times (Table 2). The growth curves of fibroblasts from the trachea, ears and lungs show a typical “S” shape (Table 3, Fig. 4). Mycoplasma test results were negative (Fig. 5). The results of H.E staining showed that the three kinds of fibroblasts grew in a spiral shape, with light blue-purple nuclei, light red cytoplasm and that long spindle-shaped or triangular cellswere typical fibroblasts in morphology (Fig. 6). Karyotype and G-banding analysis showed that the number of chromosomes in male deer fibroblasts was 2n = 70 and Bs = 5, among which there were 34 pairs of autosomes, with the morphological type of 12 submetacentric + 22 submetacentric, one pair of sex chromosomes, with the morphological type of X being metacentric, Y being st and five B chromosomes (Fig. 7a, Fig. 8a, Table 4). The number of chromosomes in female fibroblastswas 2n = 70 and Bs = 8, among which there were 34 pairs of autosomes, with morphological type was 29 submetacentric + 5 subacrocentric, one pair of sex chromosome with morphological type of X being submetacentric, and eight B chromosomes (Fig. 7b, Fig. 8b, Table 5). [Conclusion] In this study, three tissue-derived fibroblast cell lines from male and female roe deer were successfully established. When cultured in vitro, they grew well and maintained the stability of genetic stability. The karyotypes and G-banding altas of male and female roe deer chromosomes were drawn. This study provides material and basic technical support for further related research in the future.
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