Effects Of Cvtoskeleton Reconstruction Of Expression Of AQP4and Kir4.1in Rat Spinal Cord Astrocytes

Objective To detect the conditions of isolation, purification and culture of the primary spinal cord astrocytes in vitro, and to study its biological characteristics. To investigate changes of AQP4and Kir4.1expression level after cytoskeleton reconstruction of rat cortical astrocytes by different concentration of Jasplakinolide(JSK) and cytochalasin D separate treatment.Methods We extracted and cultured the primary spinal cord astrocytes from new born rats. The morphology of astrocytes was observed by employing inverted phase contrast microscope, the growth curves of them were drawn, and the protein expression of GFAP was detected by immunofluoresence. The cells were divided into control group、JSK with different concentration groups and Cyt D with different concentration groups. The cytotoxicity of JSK by different concentrations (0.05μg/ml,0.10μg/ml,0.20μg/ml,0.40μg/ml,0.80μg/ml and1.00μg/ml) was measured by MTT assay. MTT was also employed to assess survival rate of astrocytes after0.05μg/ml,0.10μg/ml,0.20μg/ml,0.40μg/ml,0.80μg/ml and1.00μg/ml of treatment for2h,12h and24h, respectively. The phalloidin and hoechest33342was combined to monitor the changes of cytoskeleton under the Laser Scanning Confocal Microscopy (LSCM). The mRNA expression levels of AQP4and Kir4.1were measured after different concentration of JSK and different concentration of Cyt D administration by real-time PCR.Results Adherent cells covered with dishes after14days culture.90%positive cells with GFAP were analyzed by immunofluoresence. MTT assay showed that the cytotoxicity of JSK gradually increased with the dosage and prolonged administration. However, there were no significant differences among0.05μg/ml,0.10μg/ml,0.20μg/ml and0.40μg/ml groups compared to control group after treatment for2h. In addition, cytochalasin D reduced survival rate of rat cortical astrocytes, but there was no statistical differences (P<0.05) among different concentrations of2h treatment group compared to control group. Morphological observation showed cytoskeleton of astrocytes was reconstructed. Cell volume was increscent, cell junction vanished. LSCM showed that JSK of different concentration could decrease cell apoptotic volume and the mRNA expression of AQP4and Kir4.1. Cytochalasin D treatment increased AQP1, AQP4, Kir4.1gene expression levelConclusion Astrocytes could be cultured and proliferated in vitro. JSK with appropriate dosage could remodel the cytoskeleton and decrease the mRNA expression of AQP4and Kir4.1. Cytochalasin D with appropriate dosage could remodel the cytoskeleton and decrease the mRNA expression of AQP1, AQP4and Kir4.lin rat spinal cord astrocytes. JSK and Cyt D can be used as candidate medicine for astrocyte edema...