| PartⅠThe expression of Id2in neuronal cells after the exposure ofhypoxia/ischemiaObjective: To investigate the expression of Id2in neuronal cells after the exposure ofhypoxia/ischemia (H/I).Methods: In vivo studies, a rat model of middle cerebral artery occlusion (MCAO)/repefusion was established, and then the expressions of Id2and Bax in neuronal cells ofthe penumbra area were examined by immunohistochemical staining, real-timequantitative polymerase chain reaction (RT-qPCR) and Western blot. Furthermore,TUNEL staining was adopted to analyze the neuronal apoptosis in the penumbra area. Invivo studies, the hypoxia-mimetic, cobalt chloride (CoCl2)-treated rat neuroblastoma B35cell line was used, and the expressions of Id2and Bax in CoCl2-treated B35cells werealso examined by RT-qPCR and Western blot. Moreover, the neuronal apoptosis wasassessed by an flow cytometric analysis for the number of cells in the sub-G1population.At last, triple immunofluorescence was used to investigate the co-localization of Id2,TUNEL and NeuN in neuronal cells of the penumbra area at24hour postMCAO/reperfusion.Results: The expressions of Id2mRNA and protein, Bax protein were up-regulatedsignificantly in neuronal cells after the exposure of H/I (P<0.01), and the extent of neuronal apoptosis elevated significantly with the longer time of H/I exposure (P<0.01).Furthermore, triple immunofluorescence demonstrated the obvious co-localization of Id2,TUNEL and NeuN in neuronal cells of the penumbra area (P<0.01).Conclusion: H/I up-regulates Id2expression in neuronal cells in vivo and in vitro, as wellas elevated neuronal apoptosis. Id2might play an important role in H/I induced neuronalapoptosis.PartⅡThe effects of Id2knock-down or over-expression on thehypoxia induced neuronal apoptosisObjective: To investigate the function of Id2in hypoxia induced neuronal apoptosis.Methods: The siRNA sequence for Id2knock-down and Lenti-virus vector pGMLV forId2over-expression were constructed, and then incorporated into the rat B35cells. WithCoCl2-treated for48hours, the apoptosis for the B35cells was assessed by an flowcytometric analysis.Results: With CoCl2-treated for48hours in B35cells, the rate of apoptosis in the siRNAgroup was (4.8±0.4)%, while the rate of apoptosis in the negative control group was(19.7±1.5)%; and the difference between the two groups on the apoptosis rate wassignificantly (P<0.01). On the other hand, the rate of apoptosis in the over-expressiongroup was (39.5±3.5)%, while the rate of apoptosis in the negative control group was(19.3±1.6)%, and the difference between the two groups on the apoptosis rate was alsosignificantly (P<0.01).Conclusion: Id2played an important role in the hypoxia induced neuronal apoptosis.Hypoxia induced neuronal apoptosis was significantly decreased after the konck-down ofId2; while the apoptosis was significantly increased after the over-expression of Id2. PartⅢThe effects and molecualr mechanisms of Id2knock-down onischemic injury of cerebral cortex in a rat model of MCAO/reperfusionObjective: To investigate the effects and possible mechanisms of siRNA-mediated Id2knock-down in a rat model of MCAO/reperfusion.Methods: After the intraventricular injection of Id2-siRNA in a rat model ofMCAO/reperfusion, the expressions of Id2mRNA, protein and Bax protein in neuronalcells of the penumbra area were examined by RT-qPCR and Western blot at24hour and72hour after MCAO/reperfusion, and the neurological status was also evaluated andscores carefully. Triphenyltertrazolium chloride (TTC) staining was used to examined theareas of ischemic infarction, and TUNEL staining was adopted to analyze the neuronalapoptosis in the penumbra area. Moreover, double immunofluorescence was used toinvestigate the co-localization of Rb-Id2and Rb-E2F1in neuronal cells of the penumbraarea.Results: Compared to the NaCl and misRNA groups, the expressions of Id2mRNA andprotein, Bax protein in neuronal cells of the penumbra area, and the neurolgocial deficitsdecreased significantly in siRNA group at24hour and72hour after MCAO/reperfusion(P<0.01). The ischemic infarction volume and the TUNEL-positive cells in penumbra areaalso decreased significantly in siRNAgroup at72hour after MCAO/reperfusion (P<0.01).At72hour after MCAO/reperfusion, the positive cells for co-localization of Rb-Id2in thepenumbra area of siRNAgroup was (91.30±7.60)/mm2, which was lower than the controlgroup (190.40±8.60)/mm2; and the difference was significantly (P<0.01). On the otherhand, the positive cells for co-localization of Rb-E2F1in the penumbra area of siRNAgroup was (220.10±9.00)/mm2, which was higher than the control group (137.30±6.50)/mm2; and the difference was also significantly (P<0.01). Conclusion: Knock-down of Id2is a therapeutic option to protect brain injury fromischemia in rats. The possible mechanisms might involve the decreased interaction ofRb-Id2and the increased interaction of Rb-E2F1, resulting in suppression of the release ofE2F1from the Rb and inactivation of genes involved in cell cycle and neuronal apoptosis. |
PDF Full Text Request