人类外周血染色体G显带的改良方法

背景:染色体G显带常规方法操作繁琐、费时、效果较差,不利于临床染色体病的检查和教学科研等。目的:寻找提高染色体G显带效果的方法。设计:观察性实验。单位:新乡医学院。材料:实验于2001-01/2005-01在新乡医学院形态实验室完成。取新乡医学院第三附属医院妇产科2001-2004年门诊不孕不育和不良生育史患者外周血共376份。RPMI1640培养液(GIBCO公司),20%小牛血清,秋水仙素,0.075mol/LKCl,甲醇,冰醋酸,胰蛋白酶(SIGMA公司),Giemsa染液。方法:改良的人类外周血染色体制备技术及G显带方法:操作步骤同常规方法,只是将部分影响因素做了调整,如秋水仙素作用时间调整到终止培养前2h,加入1mL固定液(甲醇∶冰醋酸=2∶1)进行预固定,混匀后以1500r/min离心10min。再离心后视细胞多少加新鲜固定液,制成细胞悬液,滴片。将上述玻片标本置500℃烤箱中一两小时,自然冷至37℃后,浸入胰蛋白酶液中消化3～5min左右,立即移入Giemsa染液(用1份Giemsa原液加9份pH为6.8的磷酸缓冲液配制)中染色10min,镜检计数,共观察3000分裂相数,计算标本的400～600条分裂相的百分率和分散良好百分率。染色体分散良好指染色体分散后没有重叠,数目完整,随体和着丝点明显,着色鲜明,形状清晰,且各色染色体处于同一平面上。分裂相百分率=400～600分裂相细胞数/观察细胞数×100%。主要观察指标:标本的400～600条分裂相百分率和分散良好百分率。结果:常规方法400～600条分裂相占52%,分散良好率68%;改良方法400～600条分裂相占75%,分散良好率86%,两组比较差异有显著性意义(P<0.05)。结论:改良方法获得的染色体分裂相多,分散良好,长短适中,Giemsa染色显示带纹清晰。

Modified method of chromosome G-banding in human peripheral blood

BACKGROUND: Routine methods of chromosome G-bending are complicated, time-consuming and worse in effect, which is not suitable for clinical examination and teaching investigation of chromosomal disorder.OBJECTIVE: To seek for proper method of improving the effect of chromosome G-banding.DESIGN: Observation experiment.SETTING: Xinxiang Medical University.MATERIALS: The experiment was conducted in the Laboratory of Morphous, Xinxiang Medical University between January 2001 and January 2005. 376 blood samples were obtained from patients with infertility and sterility or those had abnormal childbearing history, who came from the Clinic of Gynecology and Obstetrics, Third Hospital Affiliated to Xinxiang Medical College between 2001 and 2004. Main agents: RPMI1640 culture fluid (GIBCO Co., Ltd), 20% calf serum (CS), colchicines, 0.075 mol/L KCL, methanol, glacial acetic acid, trypase (SIGMA Co., Ltd) and Giemsa staining solution.METHODS: Modified method of human peripheral chromosomal preparation and G-banding: The procedures were the same as routine methods, while partial influential factors were regulated, for example, the action time of colchicines was set at 2 hours before the ending; 1 mL of fixation fluid (the ratio of methanol and glacial acetic acid was 2:1) was added for pre-fixation, and samples were mixed and centrifuged at 1 500 r/minute for 10 minutes. After re-centrifugalization,fresh fixation fluid was added to prepare for cell suspension and glass slide by according to the amount of cells.Above-mentioned glass samples were baked at 50 ℃ for 1-2 hours, naturally cooled to 37 ℃, digested for 3-5 minutes by immersing into trypase, rapidly stained for 10 minutes with Giemsa staining solution, and counted by the test under microscope to observe 3 000 metaphases, and the percentages; The percentage of 400-600 metaphases and well-dispersion rate were calculated. Well-dispersion rate of chromosome referred to the overlapping after dispersion with complete numbers. The trabant and kinetic body were obvious and in bright color with clear shape. Moreover, all chromosomes were on the same plane. The percentage of metaphase = the number of metaphase cells (400-600)/number of cells under observation×100%.MAIN OUTCOME MEASURES: The percentage of 400-600 pieces of metaphases in samples and good integration chromosome.RESULTS: 400-600 metaphases obtained with routine methods accounted for 52% with a well-dispersion rate of 68%,while 400-600 metaphases obtained with modified methods accounted for 75% with a well-dispersion rate of 86%, and there were significant differences between the two groups (P ＜ 0.05).the stripes are chear.CONCLUSION:Chromoseme metaphases obtained with modified methods are more in number and good in dispersion with proper size. Giemsa shows that the stripes art chear.