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穆瑶,李慧鹏,杨济洲,李云霞,曹贵方,包斯琴,李喜和.2019.中华鼢鼠成纤维细胞长期培养及其生物学特性分析.动物学杂志,54(3):395-403.
中华鼢鼠成纤维细胞长期培养及其生物学特性分析
Establishment of Long-term Culture System for Fibroblast Cells of Chinese Zokor and Analysis of Their Biological Characteristics
投稿时间:2018-07-25  修订日期:2019-04-24
DOI:10.13859/j.cjz.201903009
中文关键词:  中华鼢鼠  成纤维细胞  细胞生长曲线  核型
英文关键词:Chinese Zokor, Eospalax fontanierii  Fibroblast cells  Cell growth curve  Karyotype
基金项目:国家自然科学基金项目(No. 31560335)
作者单位E-mail
穆瑶 ① 内蒙古大学蒙古高原动物遗传资源研究中心 呼和浩特 010021② 内蒙古赛科星家畜种业与繁育生物技术研究院 呼和浩特 011517③ 内蒙古农业大学生命科学学院 呼和浩特 010018 784323771@qq.com 
李慧鹏 ① 内蒙古大学蒙古高原动物遗传资源研究中心 呼和浩特 010021② 内蒙古赛科星家畜种业与繁育生物技术研究院 呼和浩特 011517③ 内蒙古农业大学生命科学学院 呼和浩特 010018 lihuipeng0225@163.com 
杨济洲 ① 内蒙古大学蒙古高原动物遗传资源研究中心 呼和浩特 010021② 内蒙古赛科星家畜种业与繁育生物技术研究院 呼和浩特 011517③ 内蒙古农业大学生命科学学院 呼和浩特 010018 1194363746@qq.com 
李云霞 ① 内蒙古大学蒙古高原动物遗传资源研究中心 呼和浩特 010021② 内蒙古赛科星家畜种业与繁育生物技术研究院 呼和浩特 011517③ 内蒙古农业大学生命科学学院 呼和浩特 010018 liyunxia831130@163.com 
曹贵方 ① 内蒙古大学蒙古高原动物遗传资源研究中心 呼和浩特 010021② 内蒙古赛科星家畜种业与繁育生物技术研究院 呼和浩特 011517③ 内蒙古农业大学生命科学学院 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 呼和浩特 010021② 内蒙古赛科星家畜种业与繁育生物技术研究院 呼和浩特 011517③ 内蒙古农业大学生命科学学院 呼和浩特 010018 baosq@life.imu.edu.cn 
李喜和 ① 内蒙古大学蒙古高原动物遗传资源研究中心 呼和浩特 010021② 内蒙古赛科星家畜种业与繁育生物技术研究院 呼和浩特 011517③ 内蒙古农业大学生命科学学院 呼和浩特 010018 lixh@life.imu.edu.cn 
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中文摘要:
      中华鼢鼠(Eospalax fontanierii)为蒙古高原代表性动物遗传资源,本研究采集中华鼢鼠心、肝、脾、肺、肾、肌肉、气管、剑状软骨和尾尖皮肤共9种组织用于实验,其中气管、肺和剑状软骨3种组织成功建立成纤维细胞系,传了8代,并分析了这些细胞的生物学特性。主要实验方法是通过细胞计数法计算细胞的贴壁率、冻存前及复苏后的存活率、绘制细胞生长曲线;制备常规染色体标本分析染色体核型。实验结果显示,中华鼢鼠气管、肺、软骨3种组织在体外条件下培养,第3 ~ 4天出现成纤维样细胞,原代细胞分别在培养第11天、第16天、第17天,达到90%以上汇合。3种组织来源体细胞均显示成纤维细胞特征,气管成纤维细胞贴壁能力最强,24 h贴壁率最高能达到98.10%,肺成纤维细胞与剑状软骨成纤维细胞贴壁能力较弱,24 h贴壁率最高,分别为95.28%和94.88%。3种来源成纤维细胞生长曲线测定结果显示,气管成纤维细胞的增殖能力最强、肺成纤维细胞次之、剑状软骨成纤维细胞最弱。气管成纤维细胞和肺成纤维细胞在接种后的第6 ~ 7天,进入对数生长期。剑状软骨成纤维细胞在接种后第2 ~ 3天进入对数生长期。3 种成纤维细胞的生长曲线均呈“S”型,其中气管成纤维细胞增殖能力最强,在24 孔板的每个孔中,其最大增殖数目为2.435 × 104个,肺成纤维细胞最大增殖数目为1.813 × 104个,剑状软骨成纤维细胞最大增殖数目为1.521 × 104个。核型分析结果显示,中华鼢鼠的成纤维细胞染色体数目为2n = 62,30 对为常染色体,1 对为性染色体。综上所述,本研究成功建立了中华鼢鼠成纤维细胞体外培养体系,并揭示了该物种成纤维细胞的基本生物学特性,为深入研究中华鼢鼠适应低氧高二氧化碳洞道生境的分子生物学和生理学机制提供了条件,为进一步研究其遗传及物种进化提供了实验材料和参考。
英文摘要:
      At present there is no research on the establishment of Chinese zokor’s (Eospalax fontanierii) fibroblast cell lines and their biological characteristics. In this study, we selected 9 kinds of the tissues of Chinese Zokor, a representative animal genetic resource in the Mongolian Plateau, for the experiment. Fibroblast cell lines were successfully established from three tissues, including trachea, lung and xiphoid cartilage, and their biological characteristics were analyzed. The attachment rate and survival rate of the cells were calculated before cryopreservation and after resuscitation, and their chromosome karyotypes were analyzed. The fibroblast-like cells derived from primary culture of trachea, lung and xiphoid cartilage tissues were found in the third or fourth day of culture, and there was more than 90% confluence of adherent cells on the 11th, 16th and 17th day of culture, respectively. The morphology of these three kinds of somatic cells showed fibroblast characteristics (Fig. 1﹣4). The attachment ability of the trachea fibroblast cells was the greatest, and the attachment rate was 98.10% when cultured for 24 h, and the attachment ability of the lung fibroblast cells and xiphoid cartilage fibroblast cells were 95.28% and 94.88% respectively (Fig. 5﹣7). The analysis of the growth curve of fibroblast cells from three sources showed that the proliferative capacity of tracheal fibroblast cells was the best, the pulmonary fibroblast cells were the second and the xiphoid chondro fibroblast cells were the weakest. Tracheal fibroblast cells and pulmonary fibroblast cells entered the logarithmic growth stage (Fig. 8, Fig. 9) on the 6th to 7th day after inoculation. Xiphoid chondro fibroblast cells entered the logarithmic growth stage on day 2﹣3 (Fig. 10). The survival rate of cryopreserved fibroblasts from three different tissue sources decreased significantly. The growth curves of three fibroblasts are all in an “S” type. The multiplication capacity of the trachea fibroblast cells was the strongest, and the maximum proliferation number was 2.435 × 104 per well of a 24-well plate; the maximum proliferation numbers of lung fibroblast cells and xiphoid cartilage fibroblast cells were 1.813 × 104 and 1.521 × 104, respectively. The results of karyotype analysis showed that the chromosome number of fibroblast was 2n = 62 in Chinese Zokor (Fig. 11). To sum up, this study successfully established the fibroblast cells lines from Chinese zokor. The basic biological characteristics of fibroblast cells in this species were revealed, which provided basis for further studying molecular biology and physiological mechanism of Chinese zokor to adapt to the low oxygen and high carbon dioxide tunnel habitat, and provided important experimental materials and references for the further study of its heredity and species evolution.
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