【作者】 张勇；

【导师】 张立实；

【作者基本信息】 四川大学 ， 营养与食品卫生学， 2004， 硕士

【摘要】 相对于细菌和动物细胞,人的细胞用于基因突变实验有更大的意义,其结果更能客观地反映人体的情况,可避免从细菌和动物细胞所得的结果外推到人类可能产生的误差。在哺乳类细胞基因突变实验中,以TK基因突变实验和HPRT实验使用最为广泛。目前已经发展了数种可用于TK基因突变实验的TK^+/-杂合型人细胞,其中使用最多的是从WI-L2类淋巴母细胞发展起来的一系列细胞,包括TK6,TK6-E6,WTK1等。这几种细胞p53基因的状态各不相同,TK6是p53野生性,TK6-E6是p53缺失型,WTK1是p53突变型。WI-L2家族细胞目前多用于辐照的致突变实验,用以研究p53功能,在遗传毒性评价方面的应用还不广泛。 本研究选取了四种不同结构和致突变类型化学物EMS（甲磺酸乙酯,ethyl methanesulfonate）,MMS（甲磺酸甲酯,methyl methanesulfonate）,GLY（乙二醛,glyoxal）,OPD（邻苯二胺,o-phenylenediamine）做受试物,实验方法采用MLA实验非常成熟的微孔培养板法。系统比较TK6,TK6-E6两种不同p53状态细胞在TK和HPRT两个位点的基因突变实验。为WK-L2家族的人淋巴母细胞在遗传毒性检测和评价中的应用积累资料。实验结果如下: TK6和TK6-E6细胞对四种受试物的毒性耐受力不同,TK6-E6比TK6对受试物毒性较为耐受,RS（相对存活率）是较好的能真实反映毒性水平指标,但RSG（相对悬浮生长率）测定所用时间短,在剂量设计时RSG是方便和有效的选择。TK自发突变率:TK6细胞为5.48～10.72×10-6,TK6-E6为15.09～19.75×10-6,HPRT自发突变率:TK6细胞为5.33～9.55×10-6,TK6-E6细胞更多还原

【Abstract】 Human cells have species superiority over bacteria and animal cells in gene mutation assay, for the results make more sense and can be easily extrapolated to humans. The most often used loci in mammalian mutation assay are tk and hprt. There are several tk+/-genotype human cell lines were built for tk gene mutation assay and the WI-L2 lymphoblastoid family is the primary one. The major members of WI-L2 are TK6 (wild type p53), TK6-E6 (abrogated p53), and WTK1 (mutant p53); they are the same in all but the p53 status. WI-L2 family members are mainly used in ionizing radiation-induced mutation researches aimed at p53 functions at present, but seldom in the genotoxicity assay fields.In this study, four positive chemical mutagens different in structures and mutation mechanism from each other, including methyl methanesulfonate(MMS), ethyl methanesulfonate(EMS),o-phenylenediamine(OPD), glyoxal(GLY), were selected for mutation test at tk and hprt loci in human lymphoblastoid with different p53 status, TK6 and TK6-E6, respectively. And the test procedure is the same as MLA using nticrowell method. Induced mutations at tk or hprt locus in two different human lymphoblastoid cell lines by different mutagens were compared systemically to accumulate data for the further use of WI-L2 family members in mutation assay. The results are showed as below:TK6-E6 is more durable to toxicity of test agents than TK6. RS(Relative survival) is appropriate to indicating the cytotoxic, but RSG (Relative Suspension Growth) is not a bad choice with convenience and efficiency in designing dose groups, because it needs much shorter time than RS in mensuration. SMF (spontaneous mutation frequency) in TK6 at tk and hprt locus is 5.48-10.72X 10"6 and 5.33-9.55 X 10’6, respectively. SMF in TK6-E6 at tk and hprt locus is 15.09-19.75 X 10’6 and 11.76-19.78 X 10"6, respectively. All test agents induced dose-dependent mutation responses at two loci in two cell lines. The most sensitive mutation test is the test at tk locus in TK6-E6. Compared between cell lines, the mutation sensitivity is higher in TK6-E6 than that in TK6 at tk locus, but the same at hprt locus. Compared between loci, the mutation sensitivity is higher at tk locus than at hprt locus. The RSC (Rate of Slow Growth colonies) in TK6 is 0.38-0.50 and much lower than in TK6-E6 which is 0.84-0.90, RSC decreased with concentration increasing in TK6 whereas still at high level without tendential alteration in TK6-E6. RSC in both cell lines lack the ability of distinguishing agents with different mutation mechanisms.Considering aspects of cytotoxic durability, mutation sensitivity, and tumor genetics that p53 function lost at first, TK6-E6 is superior to TK6 when used in mutation assay with higher sensitivity, dosage treatment allowance and the more correlations between results and tumor generation.更多还原