基于鸡胚电转技术研究脊髓神经干细胞相关基因功能方法的建立

A method of studying the related gene function of spinal cord neural stem cells based on in ovo electroporation for chicken embryos

目的建立一种基于鸡胚电转技术研究脊髓神经干细胞（NSCs）相关基因功能的方法。方法 RT-PCR检测鸡胚发育不同时期脊髓NSCs表面标志物;在鸡胚胚龄（E）E2.5~E3时,利用活体电转基因技术将p CAGGSGFP质粒转染到鸡胚脊髓,E6时体视荧光显微镜下筛选绿色荧光蛋白（GFP）阳性胚胎,每组至少取材5个;通过脊髓横切及open-book技术观察神经纤维投射情况;普通光学显微镜下剥离出3~5条脊髓,经胰蛋白酶消化、离心后,无血清NSCs培养基重悬获得细胞铺板,于37℃、5%CO_2细胞培养箱内培养,定时观察GFP阳性脊髓NSCs的形态变化。结果 RT-PCR结果表明,鸡胚脊髓中阳性表达NSCs表面标志物;随后的脊髓横切及open-book结果表明,GFP阳性转染侧的神经纤维能穿过底板,投射到脊髓对侧;而脊髓NSCs体外培养结果显示,GFP标记的脊髓细胞具有典型的NSCs形态,继续培养后有明显突起产生。结论本实验成功建立了一种基于鸡胚电转技术研究脊髓神经干细胞相关基因功能的方法。

Objective To develop a method of studying the related gene function of the chick spinal cord neural stem cells（ NSCs） based on in ovo electroporation for chicken embryos. Methods The surface markers of the spinal cord NSCs were tested by RT-PCR at the chick different developmental stages. Subsequently,the p CAGGS-GFP plasmid was transfected into the chick spinal cord using in ovo electroporation when the fertilized eggs were cultured for E2. 5-E3; GFPpositive chick embryos were collected at E6,five samples in each group at least. The specimens were either transversely sectioned or undergone ＂open-book＂processing. The commissural axons were observed under a fluorescent microscope.Finally,the spinal cords were isolated under the stereomicroscope,and the NSCs were cultured in 37℃ and 5% CO2 condition after trypsin digest. Results RT-PCR results showed that the NSCs surface markers were expressed in chick spinal cords; the subsequent results of transverse sections and open-book processing collectively showed that GFP labeling commissural axons crossed the midline region through the floor plate to the contralateral side of the spinal cord. The GFP labeling cells isolated from spinal cords exhibited typical morphological characteristics of NSCs,and generated neuritis when cultured in vitro. Conclusion A new method is successfully established,which could be used to study the gene function of the chick spinal cord NSCs based on in ovo electroporation.