骨形成蛋白受体ⅠA在晶状体内条件敲除后对胚眼晶状体发育的影响

Influence of Bone Morphogenetic Protein ReceptorⅠA Conditional Knockout in Lens during Embryonic Eye Development

目的通过研究骨形成蛋白受体ⅠA(BMPRⅠA)基因在晶体内条件敲除小鼠中的作用,阐述胚胎发育过程中BMPRⅠA在胚眼晶状体发育中产生的影响。方法采用BMPRⅠA基因在晶体内条件敲除的小鼠alk3fl/fl;lecre和alk3fl/+;lecre,母鼠alk3fl/fl配公鼠lecre alk3fl/+或者母鼠alk3fl/+配公鼠lecre alk3fl/fl产生实验组lecre alk3fl/fl(3只),对照组为lecre alk3fl/+(3只)。提取鼠尾DNA,然后进行基因型鉴定,取胚胎固定,石蜡包埋,HE染色。接下来进行免疫荧光检查,一抗是sc-9305 bmp7,二抗是IF-0314 aG0IgG/FITC,荧光镜下观察。结果 BMPRⅠA晶体内条件敲除的小鼠与对照组比较,晶体的形态学变化差异从E13.5 d开始可以分辨,在E15.5 d已经有显著差异,但实验组和对照组均有晶体的发育。在BMPRⅠA晶体内条件敲除的小鼠中,骨形成蛋白7(BMP7)在晶体内的表达显著减少,尤其在胚胎发育的中后期(E13.5 d^E15.5 d);而对照组中BMP7的表达不受影响。结论 BMPRⅠA在晶体的发育过程中起重要的调节作用,其在晶体内条件敲除可以导致晶体发育不良,但不会导致晶体的缺失。BMP7在胚胎发育过程中表达于晶体中,而且其表达受BMPRⅠA的影响。

Objective To study the process of embryonic development from protein and molecular biology level, and investigate the function of Bone morphogenetic protein receptor ⅠA （ BMPR Ⅰ A） during development of the vertebrate eye. Methods Alk3fl/fl lecre and alk3fl/＋ lecre mice which has BMPR Ⅰ A conditional knockout in lens region were used in this experiment. 3 Cre-positive （conditional knockout, CKO） and 3 Cre-neg- ative offspring （WT） were generated by mating Cre-positive mice with Cre-negative mice. Embryos were fixed and embedded in paraffin. Sections were processed for hematoxylin and eosin staining. In immunofluorescence analysis, primary antibody sc-9305 bmp7 was used for immunofluores- cence detection. IF-0314 aGOIgG/FITC was adopted as secondary antibody. The bright field images and the fluorescent images were obtained. Re- suits Comparing the BMPR Ⅰ A lens conditional knockout mice with the control subjects, morphological difference of lens was first observed in E13.5, and significant differences were present in the E15.5 d, but both the experimental group and control group showed lens growth. In the mice of BMPRⅠ A lens conditional knockout, bone morphogenetic protein 7 （BMP7） expression was significantly reduced in lens, especially in the later pe- riod of the embryonic development （E13.5 d-E15.5 d） ;while BMP7 expression was not affected in the control group. Conclusion BMPR I A played an important regulatory role in development of lens, the loss of BMPR Ⅰ A resulted in dysplasia and morphological defects, but did not cause the lack of lens formation. BMP7 expressed in lens during embryogenesis, and its expression was influenced by BMPRⅠA.