Objective To observe the effect of Cyclin-dependent kinase5(Cdk5) konckdown by RNA interfering on astrocyte proliferation and cell cycle progression.Methods Astrocytes were cultured from SD neo nate rats and the purity of astrocytes were identified by immunofluorescence staining of glial fibrillary acidic protein.Cdk5 siRNA were designed and synthesized.Those siRNAs were tansfected into astrocytes via Lipofecttamine 2000 and the silence efficiency was counted by Real-time PCR and Western blot.Astrocytes were randomly divided into two groups:control group and Cdk5 siRNA group.At different time points (3 h,6 h,12 h and 24 h).Edu staining and flow cytometry were used to measure the proliferation and cell cycle of astrocytes.Results Specificity Cdk5 silencing on astrocytes could be established by Cdk5 siRNA sequence successfully.Edu staining showed that the percentage of Edu positive cells in Cdk5 siRNA intervention group was significantly lower than that in the control group at 3 h,6 h and 12 h after intervention (P＜0.01).flow cytometry showed that the percentage of astrocytes in S phase in Cdk5 siRNA intervention group was lower than that in the control group at 3h,6h and 12h after intervention (P＜0.05).Conclusion Knockdown of Cdk5 could inhibite the proliferation and cell cycle progression of astrocytes.It was suggested that Cdk5 might paly important roles in the proliferation of astrocytes.

Objective: To investigate the expression of Cdk5 and p35 in primary cultured astrocytes and the changes in expression induced by ischemia/reperfusion. Methods: Astrocytes were cultured from SD neonate rats. The expression and distribution of Cdk5 and p35 in cultured astrocytes were analyzed by immunofluorescence staining. Primary cultured astrocytes were subjected to oxygen-glucose deprivation/reperfusion(OGD/R),and the changes in Cdk5 and p35 expression after OGD/R were observed by Western blot. Results:Cdk5 and p35 were expressed in astrocytes,mainly in the cytoplasm and less in the nucleus,and both displayed similar cellular distribution.The expression of Cdk5 and p35 in astrocytes was increased during early phases and decreased during late phases after OGD/R. Conclusion: Both Cdk5 and p35 were expressed in astrocytes, and both showed similar, mainly cytoplasmic distribution. Cdk5 and p35 expression in astrocytes exhibited dynamic changes after OGD/R.

Objective To establish a model of cyclin-dependent kinase 5 (Cdk5) silence in astrocytes by RNA interference method. Methods A total of 240 24-hour newborn male SD rats with specific pathogen free (SPF) were enrolled, the astrocytes were cultured and identified. Three Cdk5 small interference RNA (siRNA) of Si-r-Cdk5-001 (SiT1 group), Si-r-Cdk5-002 (SiT2 group) and Si-r-Cdk5-003 (SiT3 group) were designed and synthesized, and then transfected into astrocytes via Lipofecttamine2000. The negative control group was added negative control si RNA. The transfection effect was tested by fluorescence micro-scope and the silence efficiency was counted by real-time polymerase chain reaction (PCR) and Western blot. Results The Cdk5 siRNA sequence was transfected into astrocytes successfully, and the transfection efficiency was 85 ％. The expression level of Cdk5 m RNA and protein in SiT1 group, SiT2 group and SiT3 group were significantly lower than that in control group (P ＜ 0.01). The best efficiency of silence was appeared in Si T3 group, the molecular level silence efficiency was 68 ％ and protein level silence efficiency was 79 ％. Conclusion It is demonstrated that the specific Cdk5 silencing model can be established by RNA interference technique.

Objective: To observe the gene expression of EphA receptors in the normal and ischemic hippocampus in rats. Methods: Fifty SD rats were randomly divided into two groups: sham group (n=10) and ischemia group (n=40). The Pulsinelli four-vessel occlusion model was used to induce transient global ischemia. Semi-quantitative RT-PCR was applied to measure gene expressions of EphA receptors in hippocampus at different time points (6h, 1d, 3d, 7d). To detect the cellular location of EphA4 receptors in the hippocampus,dual immunofluorescence was employed. Results: All EphA receptors including EphA1-A8 and EphA10 were expressed in normal hippocampus, and EphA4 the most abundantly expressed in normal hippocampus. EphA receptors exhibited different change pat-terns of RNA expressions in the hippocampus after transient global ischemia. EphA1, EphA2, EphA3, EphA6, EphA7 and EphA8 presented a transient up-regulation of RNA expression. However, EphA4, EphA5 and EphA10 expressed in persisted up-regulation of RNA since transient global ischemia. EphA4 also the most significantly ex-pressed in the ischemic hippocampus. Immunofluorescence study showed that EphA4 was mainly distributed in the NeuN-positive pyramidal neurons in the hippocampus, especially in the CA1-CA3 and DG regions. Conclusion:EphA receptors exhibited different patterns in RNA expression in the hippocampus after ischemia. EphA4 receptor, the most abundantly expressed in normal hippocampus, experienced dramatic change in the ischemic hippocampus. EphA4 was mainly located in the NeuN-positive pyramidal neurons in the hippocampus, especially in the CA1-CA3 and DG regions.