不同穿刺点行子宫内电转对小鼠胚胎皮层发育的影响

Influence of three puncture sites of in utero electroporation on embryonic cortex development

目的探讨子宫内电转技术操作过程中三种不同的穿刺点对胚胎死亡率、皮层厚度和皮层面积,以及细胞分化、增殖、迁移和细胞凋亡的影响。方法通过应用免疫荧光染色对Tbr1,Tbr2,Pax6,Ctip2,Caspase-3和Ki67等标记物的检测分析,评价三种不同穿刺点对细胞分化、增殖、迁移和细胞凋亡的影响。应用SPSS13.0软件使用单因素方差分析的方法对分化、增殖和凋亡实验中的细胞数量进行统计分析。结果三种不同的穿刺点无论对胚胎死亡率、皮层厚度和皮层面积,还是细胞分化、增殖、迁移和细胞凋亡的影响均无统计学意义(P>0.05)。结论三种不同的穿刺点对宫内电转胚胎无显著性影响。

Objective To compare the influence of three different puncture sites of in utero electroporation on the death rate of mice embryos, the thickness and the area of cortex, cell differentiation, cell proliferation, cell migration and cell apoptosis. Methods It groups on the bases of common puncture sites as follows： 1. electric shock only （control）; 2. injection near the posterior fontanel, at about one millimeter （here, group A）;3. injection along the antero-posterior axis into the cephalic ventricle （group B）; and 4. injection near the midpoint between the anterior fontanel and posterior fontanel, at about three millimeters （group C）. We compared the four groups using littermate mice, and repeated the experiment with 5 pregnant mice. To detect whether different injection positions affect cell differentiation, cell proliferation, cell migration and cell apoptosis, we stained cells with anti-Tbr1, anti-Tbr2, anti-Pax6 anti-Ctip2, anti- Caspase-3 and anti-Ki67. In differentiation, proliferation, and apoptosis assays, the cell number was analyzed with SPSS 13.0 software, using one way ANOVA. Results We found no statistical significant differences between the three puncture methods in the death rate of embryos, the thickness and the area of cortex, cell differentiation, cell proliferation, cell migration and cell apoptosis （P〉0.05）. Conclusion Three puncture sites used for in utero electroporation show no significantly different negative impacts during gene transfer into the embryonic mouse brain.