ABSTRACT
This study is to establish a simple and practical co-culture method of cortical neurons and astrocytes of rats. The cortex of the new-born SD rats was digested by 0.125% pancreatic enzyme, and the differential adherence was applied to obtain the mixed cell suspension of neurons and astrocytes. A low concentration of cytarabine was used to inhibit the astrocytes in a moderate way to get neuronal and astrocyte co-culture. The morphological characteristics of the cells in different times were observed under the inverted microscope. The cells began to adhere the wall 2 h after the inoculation. Neurons and astrocytes grew in a good condition under the inverted microscope 9 days after the inoculation. The results of the immunofluorescence staining and Rosenfeld's staining indicated that the co-culture of neurons and astrocytes was successful and the ratio of neurons and astrocytes was close to 1:1. A new neurons and astrocytes co-culture method, which is simple and convenient, was successfully established. It will be an efficient method for the related researches about neuronal and astrocyte co-culture in vitro.
Subject(s)
Astrocytes/cytology , Cerebral Cortex/cytology , Coculture Techniques/methods , Neurons/cytology , Primary Cell Culture/methods , Animals , Animals, Newborn , Cells, Cultured , Female , Male , Rats , Rats, Sprague-DawleyABSTRACT
Oxidized-low density lipoprotein (Ox-LDL) has been shown to play an important role in impaired surfactant metabolism and transforming growth factor-ß1 (TGF-ß1) is a critical mediator in the pathogenesis of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). In this study, we investigated whether Ox-LDL can induce TGF-ß1 protein production, and if so, how it achieves this induction in human alveolar epithelial cells (A549). We show here that Ox-LDL not only caused a dose- and time-dependent up-regulation of TGF-ß1 production, but also increased Smad3 phosphorylation, Ras/extracellular signal-regulated kinase (ERK) activity and phospholipid transfer protein (PLTP) expression in A549 cells. The inhibition of Ras/ERK activity with specific inhibitors significantly suppressed Ox-LDL-induced TGF-ß1 production, Smad3 phosphorylation and PLTP expression. Furthermore, treatment of cells with PLTP siRNA suppressed both TGF-ß1 release and Smad3 activation induced by Ox-LDL, but not the activation of Ras/ERK cascade. Taken together, we provide evidences that induction of TGF-ß1 production and Smad3 phosphorylation by Ox-LDL is mediated by Ras/ERK/PLTP pathway in human alveolar epithelial cells.
