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

1)脊柱裂高易感模型小鼠的选择：SPF室清洁后，我们制备了Wnt受体Lrp 6 KO小鼠和Pax 1 KO小鼠。我们观察了Lrp 6杂合子对交配产生脊柱裂胚胎的比例。我们发现这个比例高达40%的所有胚胎与此交配。2）药物治疗的怀孕小鼠;我们准备交配的杂合子小鼠的每一个突变和野生小鼠。我们用不同剂量和不同的治疗方案治疗卡马西平或丙戊酸。3）胚胎表型分析：通过杂合交配，获得了rs（Lrp 6亚型突变）纯合胚胎。用阿尔西安蓝和茜素染色观察软骨或骨形成的异常。在rs/rs胚胎中发现骨形成异常。我们分析了使用pQCT方法进行骨密度测量的结果。在本观察中，我们发现年轻和年老的rs/rs小鼠胫骨中的松质组织的骨密度损失。我们分析了Rankl基因在rs/rs产生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处理rs杂合子交配产生的孕鼠三天。我们检查了野生型和杂合型胚胎中脊柱裂的比例。我们无法在这一观察中发现明显的差异。

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.