Study for the development of screening method for the teratogenic medicine using spina bifida model mice

1)脊柱裂高敏感模型小鼠的选择：我们制备Wnt受体Lrp6的KO小鼠和清洁后的Pax1的KO小鼠，用于SPF房间。我们观察了Lrp6杂合对交配产生脊柱裂胚的比例。我们发现，这种交配产生的所有胚胎中，这个比例高达40%。2)妊娠小鼠的药物治疗；我们为每只突变小鼠和野生小鼠准备了杂合小鼠的交配。我们对卡马西平或丙戊酸进行了不同剂量和不同方案的治疗。我们在E12生产胚胎，并观察胚胎出现脊柱裂的比例。3)胚胎表型分析：我们还通过杂合交配获得了rs （Lrp6的次胚突变）的纯合子胚胎。用Alcianblue和茜素染色分析软骨或骨形成的异常情况。我们发现rs/rs胚胎骨形成异常。我们使用pQCT方法分析骨密度测量。在本观察中，我们发现了rs/rs小鼠胫骨松质组织的骨密度损失。我们分析了Rankl基因在rs/rs制备的成骨细胞中的表达增加。4)叶酸对脊柱裂的保护作用分析；我们用叶酸治疗由rs杂合交配产生的怀孕小鼠3天。我们比较了野生型和杂合型胚胎中脊柱裂的比例。我们在观察中没有发现明显的差异。

1)Selection of highly susceptible model mice for spina bifida : We prepare KO mice of Wnt receptor Lrp6 and KO mice of Pax1 after cleaning for SPF room. We observed the ratio of embryo having spina bifida produced with mating of Lrp6 heterozygous pair. We found this ratio was as high as 40% of all embryo produced with this mating.2)Medicine treatment of pregnant mice ; We prepare mating of heterozygous mouse for each mutant and wild mouse. We treated the carbamazepine or valproic acid with several dose and different schedule. We produced the embryo at E12 and observed the ratio of embryos showing spina bifida.3)Phenotypic analysis of embryo : We also produced homozygous embryo of rs (hypomorphic mutation of Lrp6) with heterozygous mating. The embryos were stained with Alcianblue and Alizarin for analyzing the abnormality of cartilage or bone formation. We found the abnormality of bone formation in rs/rs embryo. We analysed the bone measurement using pQCT method for bone density. In this observation we found loss of bone density at cancellous tissue in the tibia of rs/rs mouse for young and old mouse. We analysed the expression of Rankl gene in the osteoblast cells produced from rs/rs was increased.4)Analysis of protective effect of folic acid aginst spina bifida ; We treated pregnant mice produced from rs heterozygous mating with folic acid for three days. We checked the ratio of spina bifida in the embryo of wild type and heterozygous type. We could not detect clear difference in this observation.