胚胎单细胞固定技术的选择与改良

Choice and improvement of the fixation method for preimplantation genetic diagnosis by fluorescent in situ hybridization

目的:在现有数种胚胎单细胞固定方法基础上,寻求一种稳定、理想的固定技术,并进行改良。方法:以4-细胞鼠胚(A组)和人体外受精(IVF)废弃胚胎(B组)之卵裂球为材料,分别以甲醇-冰醋酸(MA)法、吐温-盐酸(TH)改良法和MA+TH结合法进行固定,比较其各自的固定率、核形态等情况。结果:3种方法的固定率以MA法最低,且3种方法固定率差异有显著性(A组3种方法P=0.00621,B组3种方法P=0.00362);A组和B组固定后得到的核形态均与固定方法有明显相关性(P值分别为0.000和7.66×10-7);信号获得率和核信号评分与固定方法无明显关联(P值分别为0.186和0.0728)。结论:TH改良法固定后核形态不良,胞质残留率高;MA法核像直径大、质量较好,但技术要求高,难于普及;MA+TH结合法则核像较大,质量最好,技术易于掌握和推广。

Objective To find an ideal fixation method for preimplantation genetic diagnosis. Methods The mouse blastomeres(group A)and the human blastomeres(group B) were fixed by a methanol/acetic acid(MA) method, an improved Tween-20/HCl (TH) method and a MA+TH combined method respectively. The fixation rate, nuclear form and signals after hybridization were observed. Results In both groups,the MA method gained the lowest fixation rate with statistical significance (P=0.00621 in group A, P=0.00362 in group B). And the nuclear form was correlated with the fixation method (P=0.000 in group A, P=7.66×10-7 in group B), while the signal gained rate and the nuclear signal score had no obvious correlation with the fixation method (P=0.186 in group A, P=0.0728 in group B). Conclusions The improved TH method obtains the poorer nuclear form than the other two methods, and the MA method requires the higher technique. The MA+TH method not only can gain the high quality nuclear form, but also is easy to learn.