| Recently,significant attention of a wide range of researchers has been toward to relationship between signaling pathway and human diseases . As a signal transducer, Activin A(ActA)has diverse roles in multiple physiological and pathological processes. Expression and neuroprotective effect has been found in many CNS degenerative diseases. However, studies of ActA/Smads pathway involved in brain ischemic insult has been poorly documented. The studies in this thesis focus on the ActA/Smads pathway in ischemic brain injury. Aim to clarify the effects and possible signal transduction mechanism, which identify novel potential target for the treatment of stroke injury.1. Establishment and assessment of focal ischemic brain injury model in ratsObjective: Establish focal ischemic brain injury model in ratMethods: Focal cerebral ischemia was induced by permanent middle cerebral artery occlusion (MCAO) in the rat. To make assessment of the model, injury and infarction volume in brain tissue was observed by HE stain and immunohistochemical stain of MAP2, neurological deficit score was tested as well as infarct volume detected at different time point after MCAO.Results: The neurological scores were significantly worse as well as the infarct volume larger in response to focal cerebral ischemic time(p < 0.05). HE stain showed infarct in the dorsolateral and lateral portions of neocortex and the entire caudoputamen in rat brains. Examination of the areas of cerebral infarction revealed loss of immunoreactivity of MAP2 in ischemic core, Swelling,fracture and abnormal arrangement of dendrite in the penumbra.Conclusions: Establish focal ischemic brain injury model in rat successfully; the filament-induced middle cerebral artery occlusion (MCAO) model correspond closely to ischemic stroke in humans.2. Expression of basic proteins in ActA/Smads signaling pathway after MCAO in ratsObjective: To investigate expression of three key components of ActA/Smads signaling pathway after various time of MCAO in rats.Methods: Focal cerebral ischemia was induced by permanent middle cerebral artery occlusion (MCAO) in the rat. We evaluated the immunohistochemical (IHC) expression of ActA, ActRIIA and Smad3 at the time point of 3h,6h,24h after MCAO. Protein expression was analyzed by detection of IOD values which were determined using Image. Pro Plus 6.0 software.Results: The expression of ActA, ActRIIA and Smad3 protein increased significantly compared to the sham group and contralateral side(p<0.05). Protein of key site in the pathway showed the same increase trend from 3h after MCAO, and reached their peak 24h after MCAO, whereas only faint expression is detected in sham group and the ipsilateral cortical areas. Staining of ActA and Smad3 was detected in the cell cytoplasm and nucleus,ActRâ…¡A in the cell cytoplasm,and in neurons only.Conclusions: Activation of ActA/Smads signaling pathway after MCAO was induced by ischemic injury, ischemic neurons in penumbra was both the source and target of ActA, the ActA autocrined by neurons act with a"target-derived"pattern in signal transduction.3. Culture and identification of neurons transformed from PC12 cells and establishment of OGD modelObjective: To culture and identified neurons transformed from PC12 cells, set up oxygen and glucose deprivation (OGD) model of activated PC12 cells.Methods: Cultured PC12 cells was activated by NGF and transformed into neurons, the neurons identified by immunofluorescence of anti NSE and MAP2. The culture were subjected to OGD injury using DMEM with no glucose and oxygen induced by NaS2O4, and were incubated in an anaerobic chamber which was refitted from a vacuum dryer.Results: Nerve growth factor (NGF) activated PC12 cells showed typical morphology of neurons, immunofluorescence identification of NSE and MAP2 was strong positive. Neuronal PC12 cells induced by NGF extended long neurites and lack the ability to divide. MTT assay and AO/EB stain showed decreased viability and increased apoptosis percentage with prolongation of exposure time.Conclusions: Neurons transformed from PC12 cells showed typical morphology and characteristic of neurons, oxygen and glucose deprivation (OGD) model of activated PC12 cells was set up by chemical and physical means successfully,which served a paradigm for isolation and purification of human ishcemic brain injury.4. Dynamic research of gene expression of different components in ActA/Smads signaling pathway of PC12 cells subjected to OGD injuryObjective: To investigate dynamic gene expression of basic components in ActA/Smads signaling pathway of neuronal PC12 cells subjected to various OGD time.Methods: ActA, ActRâ…¡A and Smad3 mRNA expression of neurons transformed from PC12 cells subjected to various OGD time were observed with Realtime PCR. Results: Real-time PCR analysis show that ActA, ActRâ…¡A and Smad3 gene expression increased significantly than control group and reached the peak after 3h of OGD, then decreased gradually(p<0.05).Conclusions: ActA/Smads signaling pathway in neurons transformed from PC12 cells was activated by OGD injury and mediated by transmembrane receptor ActRâ…¡A, showing a positive feedback mechanism in activation and signal transduction of this pathway.5. Changes in basic components expression and OGD injury correlated with blockage of transmembrane signaling transduction of ActAObjective: To investigate Changes in basic components expression and OGD injury correlated with blockage of transmembrane signaling conduction of ActA.Methods: Transmembrane signaling transduction of ActA was blocked by monoclonal antibody of ActRâ…¡A before OGD; ActA and Smad3 mRNA expression of neurons transformed from PC12 cells subjected to 3h OGD time were observed with Realtime PCR,protein expression measured with the method of westernblot; degree of OGD injury examined by Hoechst33342 staining,MTT assay and Annexin V/PI double stain FCM.Results: Real-time PCR analysis showed significant down-regulation of ActA and Smad3 gene was found by blocking the pathway at the site of ActRâ…¡A. The blockage also lowed protein expression levels of ActA and Smad3. More apoptosis of PC12 cells were also observed in the same group examined by Hoechst33342 staining,MTT assay and FCM.Conclusions: There is an activation of ActA/Smads pathway after OGD injury, and the up-regulation of ActA induced by activation of smad3 may play an foundmental role in the neuroprotective effect of ActA/Smads pathway.
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