预固定在淋巴细胞染色体制备中的量化分析
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摘要
目的深入分析预固定在淋巴细胞染色体制备中的机制及影响因素,为染色体量化制备提供依据。方法针对预固定环节中甲醇与冰醋酸的体积配比、用量及作用时间进行试验,以染色体分裂指数及分裂相合格率为指标,研究预固定环节各因素对染色体制备的影响。结果预固定液中甲醇与冰醋酸的体积配比对分裂指数影响差异无统计学意义(F=1. 549,P> 0. 05),对分裂相合格率影响差异有统计学意义(F=8. 192,P <0. 05),预固定液中甲醇与冰醋酸体积比可控制在3∶1～1∶1。预固定液用量对分裂指数(F=5. 327,P <0. 05)和分裂相合格率(F=4. 989,P <0. 05)影响差异有统计学意义,将预固定液用量维持在0. 8～0. 9 ml,染色体分散良好,无重叠。预固定作用时间对分裂指数(F=0. 821,P> 0. 05)和分裂相合格率(F=0. 458,P> 0. 05)影响差异无统计学意义,推荐预固定处理时间为1 min。结论预固定液中甲醇与冰醋酸配比控制在3∶1～1∶1之间,预固定液用量控制在0. 8～0. 9 ml之间,预固定1 min,可获得理想的染色体分裂相,将预固定环节影响因素进行量化分析,为预固定实现规范操作打下基础。
Objective To deeply research the mechanism and influencial factors of prefixation in chromosome preparation,to make a reference for quantitative chromosome preparation. Methods Aiming at the volume ratio,dosage and duration of methanol and glacial acetic acid in the pre-fixation process,the effects of various factors of pre-fixation on chromosome preparation were studied by using the chromosomal splitting index and the rate of division of the split phase. Results The proportion of methanol and glacial acetic acid had no statistically significant difference on mitotic index( F = 1. 549,P > 0. 05),but on the qualified rate of metaphase chromosome spreading( F = 8. 192,P < 0. 05). The volume ratio of methanol to glacial acetic acid in the pre-fixed solution can be controlled from 3∶1 to 1∶1. The volume of prefixative had statistically significant difference on mitotic index( F = 5. 327,P < 0. 05) and qualified rate of metaphase chromosome spreading( F = 4. 989,P < 0. 05). The dosage of the pre-fixed solution was maintained at 0. 8 ～ 0. 9 ml,and the chromosomes were well dispersed without overlap. The factor of action time had no statistically significant difference on the mitotic index( F = 0. 821,P > 0. 05) and qualified rate of metaphase chromosome spreading( F = 0. 458,P > 0. 05). The recommended pre-fixation processing time was 1 min. Conclusion The ratio of methanol and glacial acetic acid in the pre-fixed solution is under the range about 3∶1 ～ 1∶1,and the amount of pre-fixed solution is controlled between 0. 8 and 0. 9 ml. Pre-fixed for 1 min to obtain the ideal chromosome splitting phase. The quantitative analysis of the influencing factors of the fixed link lays the foundation for the pre-fixed implementation of the standard operation.
Objective To deeply research the mechanism and influencial factors of prefixation in chromosome preparation,to make a reference for quantitative chromosome preparation. Methods Aiming at the volume ratio,dosage and duration of methanol and glacial acetic acid in the pre-fixation process,the effects of various factors of pre-fixation on chromosome preparation were studied by using the chromosomal splitting index and the rate of division of the split phase. Results The proportion of methanol and glacial acetic acid had no statistically significant difference on mitotic index( F = 1. 549,P > 0. 05),but on the qualified rate of metaphase chromosome spreading( F = 8. 192,P < 0. 05). The volume ratio of methanol to glacial acetic acid in the pre-fixed solution can be controlled from 3∶1 to 1∶1. The volume of prefixative had statistically significant difference on mitotic index( F = 5. 327,P < 0. 05) and qualified rate of metaphase chromosome spreading( F = 4. 989,P < 0. 05). The dosage of the pre-fixed solution was maintained at 0. 8 ～ 0. 9 ml,and the chromosomes were well dispersed without overlap. The factor of action time had no statistically significant difference on the mitotic index( F = 0. 821,P > 0. 05) and qualified rate of metaphase chromosome spreading( F = 0. 458,P > 0. 05). The recommended pre-fixation processing time was 1 min. Conclusion The ratio of methanol and glacial acetic acid in the pre-fixed solution is under the range about 3∶1 ～ 1∶1,and the amount of pre-fixed solution is controlled between 0. 8 and 0. 9 ml. Pre-fixed for 1 min to obtain the ideal chromosome splitting phase. The quantitative analysis of the influencing factors of the fixed link lays the foundation for the pre-fixed implementation of the standard operation.
引文
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[2]中华人民共和国国家卫生和计划生育委员会.GBZ/T 248-2014放射工作人员职业健康检查外周淋巴血细胞染色体畸变检测与评价[S].北京:中国标准出版社,2014.
[3]孔祥华,韩兆东,孙大康,等.连续量化外周血淋巴细胞染色体的制备方法[J].国际遗传学杂志,2017,40(4):212-214.
[4]尹丽莎,陈杰,周军,等.滇杨根尖细胞染色体制片技术优化[J].南方农业学报,2015,46(7):1253-1258.
[5]廖亚平,张鼎,孙梓桉,等.全血培养胞质分裂阻断微核(CBMN)实验方法的改进[J].复旦学报,2014,41(3),395-399.
[6]张会勇,王文锋.用细胞膜知识探讨染色体制备过程中关键步骤的作用机理[J].生物技术世界,2015,9(11):256.
[7]赖专华,吴永平,路建超,等.外周血淋巴细胞染色体标本制片室内质量控制[J].医学动物防制,2016,32(1):112-114.
[8]HOWE B,UMRIGAR A,TSIEN F.Chromosome preparation from cultured cells[J].Journal of Visualized Experiments,2014,28(83):1-5.
[9]桂俊豪,黄国香,王铮,等.Carnoy’s预固定剂量对中期染色体分散度的影响[J].国际遗传学杂志,2006,29(6):416-419.
[10]蒋红玲,何伟佳,刘雪琴,等.外周血染色体培养方法的改良[J].世界临床医学,2016,10(6):238-240.
[11]吴子钊,程李晴,穆灵敏,等.人类外周血染色体G显带的改良方法[J].中国组织工程研究,2007,11(11):2185-2186.
[12]庄建龙,王元白,庄倩梅.外周血染色体制备方法的探讨[J].国际检验医学杂志,2015,37(17):2558-2560.
[13]谢志威,张晶,李卫凯.外周血染色体制备改良方法的应用[J].国际检验医学杂志,2013,35(1):82-83.
[14]柳华平,刘燕,曾艳,等.人外周血染色体制片技术改进研究[J].中国优生与遗传杂志,2013,21(7):67-68.
[15]李珍,赵晓平.人类外周血淋巴细胞培养的影响因素研究[J].阴山学刊(自然科学版),2016,30(1):41-43.
[16]凌攀,刘毅,蹇启政,等.从放人员染色体、微核检查的制片方法改进探讨[J].现在预防医学,2015,42(6):1096-1098.
[17]钱庆增,曲艺,王光大,等.医院放射人员染色体畸变与累积辐射剂量的关系研究[J].河北医药,2017,39(7):968-972.
[18]刘星,冯昆,张青峰,等.小鼠骨髓细胞中期染色体标本制备方法的改进[J].卫生职业教育,2015,33(18):101-102.
[19]赵淑娟,庞有志,白俊艳,等.禽类骨髓细胞染色体标本制备实验方法的改进[J].实验动物科学,2010,27(2):17-20.
[2]中华人民共和国国家卫生和计划生育委员会.GBZ/T 248-2014放射工作人员职业健康检查外周淋巴血细胞染色体畸变检测与评价[S].北京:中国标准出版社,2014.
[3]孔祥华,韩兆东,孙大康,等.连续量化外周血淋巴细胞染色体的制备方法[J].国际遗传学杂志,2017,40(4):212-214.
[4]尹丽莎,陈杰,周军,等.滇杨根尖细胞染色体制片技术优化[J].南方农业学报,2015,46(7):1253-1258.
[5]廖亚平,张鼎,孙梓桉,等.全血培养胞质分裂阻断微核(CBMN)实验方法的改进[J].复旦学报,2014,41(3),395-399.
[6]张会勇,王文锋.用细胞膜知识探讨染色体制备过程中关键步骤的作用机理[J].生物技术世界,2015,9(11):256.
[7]赖专华,吴永平,路建超,等.外周血淋巴细胞染色体标本制片室内质量控制[J].医学动物防制,2016,32(1):112-114.
[8]HOWE B,UMRIGAR A,TSIEN F.Chromosome preparation from cultured cells[J].Journal of Visualized Experiments,2014,28(83):1-5.
[9]桂俊豪,黄国香,王铮,等.Carnoy’s预固定剂量对中期染色体分散度的影响[J].国际遗传学杂志,2006,29(6):416-419.
[10]蒋红玲,何伟佳,刘雪琴,等.外周血染色体培养方法的改良[J].世界临床医学,2016,10(6):238-240.
[11]吴子钊,程李晴,穆灵敏,等.人类外周血染色体G显带的改良方法[J].中国组织工程研究,2007,11(11):2185-2186.
[12]庄建龙,王元白,庄倩梅.外周血染色体制备方法的探讨[J].国际检验医学杂志,2015,37(17):2558-2560.
[13]谢志威,张晶,李卫凯.外周血染色体制备改良方法的应用[J].国际检验医学杂志,2013,35(1):82-83.
[14]柳华平,刘燕,曾艳,等.人外周血染色体制片技术改进研究[J].中国优生与遗传杂志,2013,21(7):67-68.
[15]李珍,赵晓平.人类外周血淋巴细胞培养的影响因素研究[J].阴山学刊(自然科学版),2016,30(1):41-43.
[16]凌攀,刘毅,蹇启政,等.从放人员染色体、微核检查的制片方法改进探讨[J].现在预防医学,2015,42(6):1096-1098.
[17]钱庆增,曲艺,王光大,等.医院放射人员染色体畸变与累积辐射剂量的关系研究[J].河北医药,2017,39(7):968-972.
[18]刘星,冯昆,张青峰,等.小鼠骨髓细胞中期染色体标本制备方法的改进[J].卫生职业教育,2015,33(18):101-102.
[19]赵淑娟,庞有志,白俊艳,等.禽类骨髓细胞染色体标本制备实验方法的改进[J].实验动物科学,2010,27(2):17-20.
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