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Thesis Advisor白国栋 ; 蒋一珪
Degree Grantor中国科学院水生生物研究所
Place of Conferral中国科学院水生生物研究所
Degree Discipline遗传学
Abstract本文选用草鱼(Ctenopharyngodon idellus)作为实验鱼,首先确立了一草鱼尾鳍组织的二倍体细胞系,GCTF-2。该细胞系用TC-199培养液加15%小牛血清培养。细胞生长旺盛,最高分裂指数达30.04‰。细胞培养5天,细胞数增加5倍。现已培养十七个月,传代100次。细胞在28 ℃生长良好,在20 ℃-35 ℃也可生长。培养到第90代,染色体数2n = 48的细胞占总细胞数的87.6%,因此确立为二倍体细胞系。利用姐妹染色单体分化染色技术初步确定该细胞系细胞的有丝分裂周期时间为21-33小时,该细胞系对草鱼呼肠弧病毒(GCRV)敏感。然后,利用建系的草鱼细胞和草鱼体内肾细胞,以姐妹染色单体交换为指标,首次在鱼类确立了一个敏感的检测化学诱变或致癌物质的综合试验系统。GCTF-2细胞的自发姐妹染色单体交换频率为每细胞2.85 ± 1.089 SCEs; 草鱼肾细胞(体内)r 基线姐妹染色单体交换频率为每细胞3.05 ± 2.523 SCEs。化合物丝裂霉素C(MMC)亚硝基胍(MNNG),农药叶蝉散(MIPC)和三唑酮(Bayleton)均能诱发GCTF-2细胞SCE频率显著升高。MMC和MNNG能诱发草鱼肾细胞(体内)SCE频率显著升高。农药叶蝉散诱发草鱼肾细胞的SCE频率与对照组无显著差异。MMC在一定的剂量水平对草鱼肾细胞姐妹染色单体分化(SCD)频率没有影响。MMC和三唑酮在一定的剂量水平对GCTF-2细胞周期动力学有一定的影响。
Other AbstractGrass Carp(Ctenopharyngodon idellus) was chosen as a experimental fish in the present and the results which were obtained from it are as following: First, a diploid fish cell line, GCTF-2, from Grass Carp tail fin was established. The line was cultivated in TC-199 medium with 15% fetal calf serum containing a moderate amount of antibiotics. The cells can increase five times in five days and the cell morphology is a complex of epithelial-like cell and fibroblasticlike cell. The highest index of mitotic division of GCTF-2 is 3.004% in 48 hours after plating. The optimal grown temperature is 28 ℃, though the cells will grow at temperature from 20-35 ℃. A interesting discovery we did was that the cells which had been kept in the incubator at 20 ℃ for two months without any care could grow normally after plating. At 90 generation, the cells which had a normal number of chromosomes of Grass Carp (2n = 48) accounted for 87.6%, so the line was determined as a diploid fish cell line. By the end of January 1985, the line had been maintained in proliferation for over 17 months through 100 generations. Using the techniques of BrdU-labling and sister chromatid differential staining, we have estimated the cell cycle time of mitotic division of CGTF-2 between 21-33 hours. GCTF-2 was observed to be susceptible infection with Grass Carp Reovirus(GCRV). Second, utilizing the technique of sister chromatid differential staining and considering the sister chromatid exchange frequency as a indicator, we have investigated the spontaneous SCE frequency of GCTF-2 cells and the baseline SCE frequency of Grass Carp renal cells (in vivo) and the possible mutagen activities of 4 chemicals (mitomycin C, MMC; N-methyl-N'-nitro-N-nitrosoguanidine, MNNG; MIPC; and Bayleton) in the cultures of GCTF-2 diploid cells and Grass Carp renal cells. The experimental results indicated that the spontaneous SCE frequencies of GCTF-2 and the baseline SCE frequencies of Grass Carp renal cell are low and there is not significat differentiation between them; All 4 chemicals can induce significant increase of SCE frequency of GCTF-2 cells at the concentrations we used. MMC and MNNG can induce remarkable increase of SCE frequencies of Grass Carp renal cells, but MIPC can not. In addition, MMC and Bayleton have a activity of cell-cycle deley on GCTF-2 cells, and MMC has no effect on frequency of SCD in renal cells of Grass Carp in vivo at the concentrations we used. From above, using the indicator of SCE, we can firstly establish a susceptible test system measuring chemical mutagen and carcinogenic substances in vitro and in vivo in fish, and it may be considered that MIPC and Bayleton are two relatively safe pesticides.
Document Type学位论文
Recommended Citation
GB/T 7714
魏彦章. 鱼类体外体内姐妹染色单体交换频率的分析和应用[D]. 中国科学院水生生物研究所. 中国科学院水生生物研究所,1985.
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