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为了建立一种能够获得高纯度成年大鼠海马神经前体细胞(HPCs)的体外贴壁培养方法,并鉴定HPCs上是否存在功能性L-型钙通道,分离Wistar成年大鼠海马组织,制成单细胞悬液,利用无血清培养技术,在添加碱性成纤维细胞生长因子(bFGF)、表皮生长因子(EGF)、N2和B27supplement的DMEM/F12培养液中进行培养.连续传代,采用细胞免疫荧光法对第六代细胞进行鉴定,呈巢蛋白(nestin)阳性的细胞可达99.9%.把培养高纯度的细胞在分化培养基中诱导分化14天后,表现为神经元和星形胶质细胞的形态,且分别呈Ⅲ型β-微管蛋白(Tuj1)阳性和胶质纤维酸性蛋白(GFAP)阳性.细胞免疫荧光和免疫印迹结果显示,HPCs表达L-型钙通道的Cav1.2α1C和Cav1.3α1D亚单位,共聚焦钙成像证明了功能性L-型钙通道的存在,并且利用全细胞膜片钳技术记录到了L-型钙电流.以上结果表明成年Wistar大鼠的海马HPCs可表达功能性的L-型钙通道.
In order to establish a method for adherent culture of hippocampal neural progenitor cells (HPCs) with high purity in adult rats and to identify the existence of functional L-type calcium channels on HPCs, the Wistar adult rat hippocampus tissue was isolated and made into Single cell suspension was cultured in DMEM / F12 medium supplemented with basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), N2 and B27supplement using serum-free culture technique.Continuous passage, using cellular immunity Sixth generation cells were identified by fluorescence method, and the number of nestin positive cells was 99.9%. After differentiation of cultured high purity cells in differentiation medium for 14 days, they showed neurons and astrocytes (Tuj1) positive and glial fibrillary acidic protein (GFAP) positive.Results of immunofluorescence and Western blot showed that HPCs expressed Cav1.2α1C and Cav1 .3α1D subunit, confocal calcium imaging demonstrated the presence of a functional L-type calcium channel, and L-type calcium currents were recorded using whole-cell patch clamp techniques.These results indicate that hippocampal HPCs in adult Wistar rats express functional L - type calcium channel.