单个卵裂球荧光原位杂交标本前处理的研究

Research on the Pre-treatment Technique for Single Blastomere before Fluorescence in Situ Hybridization

目的建立一种简单有效的单细胞荧光原位杂交前单个细胞的定位和查找技术,促进胚胎种植前遗传学诊断方法的应用。方法卵裂球在玻片上固定后被随机分为2组,一组直接应用FISH检测X、Y染色体;另一组先用瑞氏染色法染片,在荧光显微镜下查找单个细胞,乙醇脱色,再进行FISH杂交,杂交后查找X、Y染色体信号。比较两种方法核和荧光信号检出率。结果直接查找法和瑞氏染色定标法核的检出率分别为81.3%(39/45)和83.3%(45/54),两者核检出率的差异无显著性;信号检出率分别为70.8%(34/45)和74.1%(40/54),两者检出率的差异无显著性。结论瑞氏染色定标法在单细胞荧光原位杂交前标本的查找中简单、方便可行。

Objective To set up a simple and effective method to lookup single blastomeres on a slide before fluorescence in situ hybridization so that preimplantation genetic diagnosis （PGD）teehnique could be used widely. Methods The blastomeres fixed onto microscope slides were divided randomly into two groups. In group one slides were processed for hybridization without any additional treatment,in group two the blastomeres fixed onto microscope slides were strained by Wright＇s stain firstly. Then to lookup the blastomeres and record its coordinate under microscope, immediately fade Wright＇s stain by using ethanol. FISH signals of chromosome X, Y were observed on a slide at the same coordinate after hybridization. The two methods were compared to evaluate efficiency of fixation and the intensities of FISH signals. Results In group one,39（81.3%）of 45 blastomeres were found,while 45（83.3%）of 54 were found in group two. There was not a significant difference in the signal gained rate between the two groups. Conclusions Our results indicate that Wright＇s stain is simple and available method to checkup single blastomere before fluorescence in situ hybridization.