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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 onin 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pCAGGS-GFP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%CO2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 onin 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 pCAGGS-GFP plasmid was transfected into the chick spinal cord using in ovoelectroporation when the fertilized eggs were cultured for E2.5-E3; GFP-positive 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 onin ovo electroporation.
［1］Reynolds BA, Weiss S. Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system［J］. Science, 1992,255(5052):1707-1710.
［2］Vaccarino FM, Ganat Y, Zhang Y, et al. Stem cells in neurodevelopment and plasticity［J］. Neuropsychopharmacology, 2001,25(6):805-815.
［3］Muramatsu T, Nakamura A, Park HM. In vivo electroporation: a powerful and convenient means of nonviral gene transfer to tissues of living animals［J］. Int J Med, 1998,1(1):55-62.
［4］Croteau LP, Kania A. Optimisation of in ovo electroporation of the chick neural tube［J］. J Neurosci Methods, 2011, 201(2):381-384.
［5］Gao Y, Bai C, Wang K, et al. All-trans retinoic acid promotes nerve cell differentiation of yolk sac-derived mesenchymal stem cells［J］. Appl Biochem Biotechnol, 2014, 174(2):682-692.
［6］Yang CQ, Mao HL, Lin JT. Establishment of chicken embryo culture system and in vivo electroporation methods［J］. Acta Anatomica Sinica, 2012, 43(4):564-568. (in Chinese)
杨慈清，毛会丽，林俊堂.鸡胚培养及活体电转基因方法的建立［J］.解剖学报，2012，43(4)：564-568.
［7］Hu AZh, Yang CQ, Fu SL, et al. A new method to study nerve fiber projecting in the spinal cord during chicken embryo development ［J］.Acta Anatomica Sinica, 2014,45(5):598-602.(in Chinese)
胡阿珍，杨慈清，付苏雷，等. 一种鸡胚发育过程脊髓神经纤维投射研究方法的建立［J］.解剖学报，2014,45(5)：598-602.
［8］Gandy S, Zhang YW, Ikin A, et al. Alzheimer’s presenilin 1 modulates sorting of APP and its carboxylterminal fragments in cerebral neurons in vivo［J］. J Neurochem, 2007,103(2):619-626
［9］Zhang XY, Luo Q, Zhang JH,et al. Optimization of electroporation parameters for SD rats neural stem cells culture in suspension［J］. China Joumal of Modern Medicine, 2012,22(23):27-30. (in Chinese)
张晓英，罗琼，张季红，等.电穿孔转染SD大鼠神经干细胞条件优化［J］.中国现代医学杂志，2012，22(23)：27-30.
［10］Liu W. The techniques and methods of gene function studying by chick embryo model［J］. China Poultry, 2009, 31(12):7-10. (in Chinese)
刘薇.利用鸡胚模型进行基因功能研究的技术和方法［J］.中国家禽，2009,31(12)：7-10.
［11］Jaroszeski MJ, Dang V, Pottinger C, et al. Toxicity of anticancer agents mediated by electroporation in vitro［J］. Anticancer Drugs, 2000,11(3):201-208.
［12］Rashidi H, Sottile V. The chick embryo:hatching a model for contemporary biomedical research［J］. Bioessays, 2009,31(4):455-465.
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