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郭晶营,韩红梅,梁延峰,范丽红,王子馨,赵丽霞,张金吨,李云霞,李喜和.2018.梅花鹿体细胞航天诱变后的生物学特性变化分析.动物学杂志,53(2):255-263.
梅花鹿体细胞航天诱变后的生物学特性变化分析
Biological Characteristic Changes of Somatic Cells Following Space Mutagenesis in Cervus nippon
投稿时间:2017-08-11  修订日期:2018-03-02
DOI:10.13859/j.cjz.201802011
中文关键词:  航天诱变  梅花鹿体细胞  生长曲线  核型
英文关键词:Space-mutagenesis  Cervus nippon somatic cell  Growth curve  Karyotype
基金项目:蒙古高原动物遗传资源库建立与利用项目内蒙古重点科技项目(No. 20130216)
作者单位E-mail
郭晶营 内蒙古大学蒙古高原动物遗传资源研究中心 1027037223@qq.com 
韩红梅 内蒙古大学蒙古高原动物遗传资源研究中心内蒙古赛科星家畜种业与繁育生物技术研究院有限公司 hongmei1120@163.com 
梁延峰 内蒙古赛科星家畜种业与繁育生物技术研究院有限公司 670985991@qq.com 
范丽红 内蒙古赛科星家畜种业与繁育生物技术研究院有限公司 395831676@qq.com 
王子馨 内蒙古赛科星家畜种业与繁育生物技术研究院有限公司 wangzixin123456@163.com 
赵丽霞 内蒙古大学 蒙古高原动物遗传资源研究中心内蒙古赛科星家畜种业与繁育生物技术研究院有限公司 zhaolixia0814@163.com 
张金吨 内蒙古大学蒙古高原动物遗传资源研究中心内蒙古赛科星家畜种业与繁育生物技术研究院有限公司 zhang-jindun@hotmail.com 
李云霞 内蒙古赛科星家畜种业与繁育生物技术研究院有限公司 liyunxia831130@163.com 
李喜和 内蒙古大学 蒙古高原动物遗传资源研究中心内蒙古赛科星家畜种业与繁育生物技术研究院有限公司 lixh@life.imu.edu.cn 
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中文摘要:
      本研究以自主研发的聚偏二氟乙烯膜(PVDF)细胞培养技术进行了梅花鹿(Cervus nippon)体细胞的航天培养及生物诱变,并分析了其细胞在航天诱变后的生长曲线和核型特性。采用耳缘皮肤组织块贴壁培养的方法进行体细胞的原代培养;胰蛋白酶消化的方法完成细胞的传代培养;依照程序降温的方法完成细胞的冷冻保存;每天计数24孔板每孔的活细胞数并利用Excel 97-2003绘制出细胞的生长曲线,采用IBM SPSS Statistics 23统计软件回归分析程序中的非线性回归分析子程序Logistic曲线模型分析细胞生长曲线的拟合度;以常规染色体标本制备技术,利用细胞遗传工作站(AI)软件对梅花鹿体细胞的染色体核型进行分析。实验结果表明,新开发的PVDF膜细胞培养技术可有效保持梅花鹿体细胞在太空飞行过程中的存活并成功回收、传代建系。通过细胞传代培养观察发现,该体细胞经航天诱变后保持正常的成纤维细胞特征,生长曲线呈“S”型,航天诱变组体细胞在培养4 d进入对数生长期,与对照组体细胞在培养2 d进入对数生长期相比其细胞增殖速度减慢。拟合生长曲线分析表明,航天诱变组细胞生长拟合度值与对照组相似,但细胞增殖拐点时间、拐点细胞量分析结果证明,航天诱变后的体细胞增殖速度减慢。核型分析显示,航天诱变梅花鹿体细胞染色体数保持2n = 66,染色体形态正常,核型为66(XX),其中32对常染色体,1对性染色体。本研究建立了哺乳动物在航天运行条件下的细胞培养PVDF新技术,并分析了航天诱变对于梅花鹿细胞生物学特性变化的影响,为以后进行相关研究提供了基础信息和技术支持。
英文摘要:
      In this study, we for the first time carried out Sika Deer (Cervus nippon) somatic cell culture in the space by using self-developed olyvinylidene fluoride (PVDF) membrane culture method, and analyzed the biological characteristic changes of these cells, including cell growth curve and karyotype characteristics. Ear skin tissue was collected for primary adherent culture of somatic cells, and these cells were then subcultured after trypsin digestion. The cells were cryopreserved by using a traditional procedure cooling system. The cell number was counted every day in the 24-well plates and the cell growth curve was made by applying Excel 97-2003. Non-linear regression analysis subroutine Logistic curve model from the IBM SPSS Statistics 23 statistical software was used to analyze growth curve fitting degree, and normal chromosome specimen preparation technology and cytogenetic workstation (AI) software were used to analyze chromosome karyotype of space-treated cells. The results showed that new technology of developed PVDF membrane for cell culture (Fig. 1) could effectively maintain the survival of the C. nippon somatic cells in the space and the cells successfully proliferated after recovery. The somatic cells were still showing normal fibroblast morphology after the space-mutagenesis (Fig. 2). The growth curve of somatic cells showed “S” model which was similar to that of control somatic cells. The space-treated group of cultured somatic cells entered the logarithmic growth phase by 4 days after the cultivation, while it took 2 days in the control group (Fig. 3). The rates of fitting growth curve were similar between two the groups, however, the cell inflexion time and cell volume of somatic cells were significantly changed in space-treated cells (Table 1). Karyotype analysis showed that the chromosome number was still 2n = 66 (XX), and chromosome morphology was normal, 32 of which were autosomes and one pair of sex chromosome (Fig. 4). This study established a new technology of somatic cell culture for domestic animals in the space condition, and also analyzed the effect of the space conditions on biological characteristics of C. nippon somatic cells, which provided some basic and important information and technique for future studies of animal genetics.
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