Neuronal differentiation and growth control of neuro-2a cells after retroviral gene delivery of connexin43
Neuronal differentiation and growth control of neuro-2a cells after retroviral gene delivery of connexin43
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DOI：
10.1074/jbc.m003917200
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发表时间：
2000-11-03
影响因子：
4.8
通讯作者：
Naus, CCG
中科院分区：
文献类型：
--
作者：
Mao, AJ;Bechberger, J;Naus, CCG
鉴于间隙连接细胞间通讯在神经元分化和生长控制中的作用，我们检查了神经母细胞瘤细胞系中连接蛋白 43 (Cx43) 表达的影响。水泡性口炎病毒 G 蛋白 (VSVG) 假型逆转录载体被设计为在通讯缺陷的 Neuro-2a (N2a) 细胞系中共表达绿色荧光蛋白 (GFP) 和 Cx43。 293 GPG 包装细胞系用于产生编码 GFP、Cx43 或嵌合 Cx43 GFP 融合蛋白的 VSVG 假型逆转录载体。通过流式细胞术测量 GFP 荧光，病毒上清液的滴度约为 2.0 x 10(7) 菌落形式单位 (CFU)/ml，并且不含具有复制能力的逆转录病毒。用视黄酸 (20 muM) 处理 7 天后，N2a 转化体（N2a-Cx43 和 N2a-Cx43 GFP）维持了 Cx43 和 Cx43 GFP 的表达。电生理学和染料注射分析证明，两种构建体的表达导致功能耦合。细胞生长的抑制与 Cx43 或 Cx43 GFP 的表达和视黄酸处理相关。根据神经丝的形态学和免疫细胞化学，与表达 Cx43 构建体的细胞相比，N2a 细胞的分化没有观察到差异。总之，N2a 细胞中 Cx43 的组成型表达不会改变视黄酸诱导的神经元分化，但会增强生长抑制。
Given the roles proposed for gap junctional intercellular communication in neuronal differentiation and growth control, we examined the effects of connexin43 (Cx43) expression in a neuroblastoma cell line. A vesicular stomatitis virus G protein (VSVG)-pseudotyped retrovector was engineered to co-express the green fluorescent protein (GFP) and Cx43 in the communication-deficient neuro-2a (N2a) cell line. The 293 GPG packaging cell line was used to produce VSVG-pseudotyped retrovectors coding for GFP, Cx43, or chimeric Cx43 GFP fusion protein. The titer of viral supernatant, as measured by flow cytometry for GFP fluorescence, was approximately 2.0 x 10(7) colony form units (CFU)/ml and was free of replication-competent retroviruses. After a 7-day treatment with retinoic acid (20 muM), N2a transformants (N2a-Cx43 and N2a-Cx43 GFP) maintained the expression of Cx43 and Cx43 GFP. Expression of both constructs resulted in functional coupling, as evidenced by electrophysiological and dye-injection analysis. Suppression of cell growth correlated with expression of both Cx43 or Cx43 GFP and retinoic acid treatment. Based on morphology and immunocytochemistry for neurofilament, no difference was observed in the differentiation of N2a cells compared with cells expressing Cx43 constructs. In conclusion, constitutive expression of Cx43 in N2a cells does not alter retinoic acid-induced neuronal differentiation but does enhance growth inhibition.