文献类型：期刊文献

中文题名：四个达摩凤蝶新孵幼虫细胞系的建立及其生物学特性

英文题名：Establishment and characterization of four cell lines developed from Papilio demoleus( Lepidoptera: Papilionidae)

作者：丁伟峰[1] 冯颖[1] 张欣[1] 李娴[1] 王成业[1] 谢世聪[1]

第一作者：丁伟峰

机构：[1]中国林业科学研究院资源昆虫研究所国家林业局资源昆虫培育与利用重点实验室

年份：2015

卷号：58

期号：8

起止页码：826-835

中文期刊名：昆虫学报

外文期刊名：Acta Entomologica Sinica

收录：CSTPCD;;Scopus;北大核心:【北大核心2014】；CSCD:【CSCD2015_2016】；

基金：国家林业局引进国际先进林业科学技术("948")项目(2013-4-53);中央及公益性科研院所基本科研业务费专项资金(riricaf2012003M)

语种：中文

中文关键词：达摩凤蝶;新孵幼虫;昆虫细胞系;分泌型碱性磷酸酶;重组杆状病毒

外文关键词：Papilio demoleus; neonate larvae; insect cell line; secreted alkaline phosphatase; recombinant baculovirus

分类号：Q965.8

摘要：[目的]建立达摩凤蝶Papilio demoleus Linnaeus新孵幼虫细胞系,并对其生物学特性进行研究。[方法]使用改良配方的Grace培养基并辅以20%胎牛血清,通过原代和传代培养建立达摩凤蝶新孵幼虫细胞系。通过显微观察、细胞活力分析、核型分析和分子鉴定,获得4个新建细胞系的生物学特性数据。使用Bac-To-Bac杆状病毒表达系统构建重组分泌型碱性磷酸酶杆状病毒（Ac MNPV-SEAP）。使用有限稀释法测定达摩凤蝶细胞系对Ac MNPVSEAP的半数组织培养感染剂量（TCID50）,比较达摩凤蝶细胞系对Ac MNPV-SEAP的敏感性。[结果]建立了4个贴壁生长的达摩凤蝶新孵幼虫细胞系（RIRI-Pa De-1,RIRI-Pa De-2,RIRI-Pa De-3及RIRI-Pa De-4）,且均传至60代以上。形态学方面,细胞系RIRI-Pa De-1和RIRI-Pa De-4较为相近,均由圆形、梭形以及多边形细胞组成,细胞系RIRIPa De-2主要为圆形细胞,而RIRI-Pa De-3主要为类似表皮细胞和纤维状细胞。细胞增殖动力学方面,4个细胞系的群体倍增时间分别为69.77,67.42,81.48及65.43 h。核型分析显示4个细胞系染色体数量均呈正态分布,其中RIRI-Pa De-2,RIRI-Pa De-3以及RIRI-Pa De-4的染色体数目分布区间比较接近,在45～97条之间,RIRI-Pa De-1的染色体条数相比偏少,在36～90条之间。通过比对4个达摩凤蝶细胞系和虫卵的细胞色素c氧化酶亚基I（COI）基因序列,证明4个新建细胞系来源于达摩凤蝶组织。比较4个细胞系对Ac MNPV-SEAP敏感性发现RIRI-Pa De-3对此病毒较为敏感,可作为重组杆状病毒表达的宿主。[结论]虽然4个新建达摩凤蝶细胞系的来源相同,具有相同的遗传背景,但其生物学特征仍有明显差异,具有进一步研究的价值。
[Aim]This study aims to establish and characterize cell lines developed from the neonate larvae of Papilio demoleus Linnaeus. [Methods]Modified Grace＇s medium supplemented with 20% fetal bovine serum was used to culture tissues from neonate larvae of P. demoleus. The methods of cell morphology analysis,growth analysis,chromosome analysis,and molecular identification of the cell lines were used to obtain the biological characteristics of new cell lines. In order to study the viral susceptibility of four cell lines derived from P. demoleus,recombinant baculovirus carrying secreted alkaline phosphatase（ Ac MNPV-SEAP） was constructed using Bac-to-Bacbaculovirus expression system to infect the cell lines. The median tissue culture infectious dose（ TCID50） of each cell line was measured using limiting dilution method by measuring the expression of recombinant SEAP. [Results]Four new cell lines developed from the neonate larvae of P. demoleus were established on modified Grace＇s medium and named RIRI-Pa De-1,RIRI-Pa De-2,RIRI-Pa De-3 and RIRI-Pa De-4,respectively. These cells were subcultured for approximately 60 passages. There were significant differences in biological characteristics between different cell lines. According to morphology,RIRI-Pa De-1 and RIRI-Pa De-4were composed of round,spindle and polygonal cells,RIRI-Pa De-2 was mainly composed of round cells,and RIRI-Pa De-4 was mainly composed of epidermal-like and fibrous cells. The cell population doubling time（ PDT） of IRI-Pa De-1,RIRI-Pa De-2,RIRI-Pa De-3 and RIRI-Pa De-4 was 69. 77,67. 42,81. 48 and 65. 43 h,respectively. Karyotype analysis showed that the number of chromosome of each cell line fitted the normal distribution. The chromosome numbers of RIRI-Pa De-2,RIRI-Pa De-3 and RIRI-Pa De-4varied widely from 45 to 97,while that of RIRI-Pa De-1 varied from 36 to 90. All of the four cell lines could be infected with the Ac MNPV-SEAP,but RIRI-Pa De-3 was more susceptible to Ac MNPV-SEAP.[Conclusion]Although the four new cell lines developed from the neonate larvae of P. demoleus have the same genetic background analyzed by DNA amplification fingerprinting（ DAF） using mitochondrial cytochrome c oxidase subunit I（ COI） gene,there are still significant differences in the basic biological characteristics among them. It is worthy to be further researched.