一种改良方法构建大鼠脊髓损伤体外细胞模型

Construction of in vitro cell model of spinal cord injury in rats with a modified method

目的探讨大鼠脊髓神经细胞原代培养、鉴定的方法并构建大鼠脊髓损伤体外细胞模型。方法取孕15 d SD大鼠胚胎,分离脊髓组织并采用改良的胰酶消化联合机械分离法进行脊髓神经细胞的原代培养;采用免疫荧光三标染色鉴定脊髓神经细胞;应用H_(2)O_(2)干预构建大鼠脊髓损伤体外细胞模型。结果取孕15 d大鼠胚胎采用改良的胰酶消化联合机械分离法成功分离培养原代脊髓神经细胞。免疫荧光染色可见特异的NeuN及β tubulin-Ⅲ染色,符合脊髓神经细胞的特征,经鉴定脊髓神经细胞纯度为90%。应用H_(2)O_(2)干预大鼠原代脊髓神经细胞建立脊髓损伤体外细胞模型时以干预浓度700μM,时间3 h最为合适。结论采用改良的胰酶消化联合机械分离法分离培养大鼠脊髓原代神经细胞,细胞产量高、活性好,纯度可以达到90%;采用H_(2)O_(2)干预大鼠脊髓原代神经细胞以建立脊髓损伤体外细胞模型可靠性高,重复性好。

Objective To culture and identify the spinal primary neurocytes and construct the in vitro cell model of spinal cord injury(SCI) in rats.MethodsRat embryos of 15 days were used to isolate spinal cord tissues and culture primary spinal neurocytes with modified trypsin digestion combined with mechanical separation method. Immunofluorescence staining(Neu N/β tubulin-Ⅲ/Hoechst 33258) was used to identify spinal neurocytes. Hydrogen peroxide(H_(2)O_(2)) was used to construct the in vitro cell model of SCI in rats.ResultsPrimary spinal neurocytes were isolated and cultured from 15-day pregnant rat embryos by modified trypsin digestion and mechanical separation. Fetal rat spinal cord was easy to peel off and had strong survival ability in vitro. Specific NeuN and tubulin-Ⅲ staining were observed by immunofluorescence staining, which was consistent with the characteristics of spinal neurocytes. The purity of spinal neurocytes was identified to be 90 %. The cell model of SCI in vitro was constructed by H_(2)O_(2) intervention of primary spinal neurocytes in rats, and the concentration of H_(2)O_(2) intervention was 700 μM and the time was 3 h by CCK-8 method.ConclusionThe primary spinal neurocytes are isolated and cultured by modified trypsin digestion combined with mechanical separation, with high yield, good viability and purity up to 90 %. H_(2)O_(2)-interfered rat primary spinal neurocytes to construct the in vitro cell model of SCI in rats have high reliability and good repeatability.