The Effect Of PTEN Down-regulation On Learning And Memory Of Rats With Vascular Dementia And Its Action Mechanis

Objective: Vascular dementia (VD) is one of main types of senile dementia, with a syndrome of acquired intelligence capacity damage coming from mental disorder caused by various kinds of cerebrovascular diseases. It's mainly clinical manifestation is remembrance and cognition functional impairment. Thus the focal point and breakthrough on the study of VD is to improve its learning and memory function effectively. The tumor suppressor PTEN (phosphatase and tensin homology deleted on chromosome 10) is a lipid and protein phosphatase. Reseaches have found that down-regulation of PTEN has protective effect on regulating diverse neuronal function. As a key negative regulator of the PI3K/Akt signaling pathway, PTEN also plays an important role in the neuronal differentiation, dendritic branching and synaptic plasticity. Therefore, down-regulating the expression of PTEN may involved in the improvement of learning and memory of VD rats. In this study, in order to provide a therapeutic approach to promote recovery from vascular dementia, we will assess the contribution of PTEN to mediate pathological changes, CREB expression in hippocampus and the improvement of learning and memory by adenovirus-mediated RNAi in VD rats model.Methods:1.The recombinated adenovious was amplified in 293 cells(human embroyonic kidney cells)and purified by Sartorius Vivapure AdenoPACK 20 kit , then determined the virus titer.2. Appropriate titers of recombinated adenovious and adenovious preservation solution (control group) were used to transfect into the hippocampus cell layer of SD rats. To explore the transfection efficiency of adenovirus and inhibition efficiency of RNAi, fluorescence microscope was applied to observe the red fluorescent protein (RFP) expression, at the same time, reverse transcriptionpolymerases chain reaction (RT-PCR) and western blotting method were used to determine the changes of PTEN expression from mRNA and protein levels.3. Forty male SD rats were randomly divided into sham group (sham) and VD model groups (VD),adenovious injection groups (siPTEN), negative control groups (AdRFP). Two-vessel occlusion (2VO) was employed to make vascular dementia. Four weeks later, morris water-maze test was performed to study the ability of spatial learning and memory in each group of rats. HE staining and Nissl staining examination were applied to detect the histopathological changes.4. To investigate the effects of PTEN down-regulation on the expression of CREB in the region of hippocampus neurons from VD rats, RT-PCR and immunohistochemistry were used to determine the changes of Akt,CREB expression form mRNA and protein levels. Thus, it may be helpful to study the molecular mechanism of PTEN gene improving learning and memory in VD rats.Results:1. After transfection, the red fluorescence was observed in 293 cells. The titer of amplified recombined adenovirus AdR-siPTEN and AdRFP were 3.36×1010 pfu/ml, 2.98×1010pfu/ml respectively.2. Positive expression of transgene was found in hippocampus after the transfection of adenovirus, RFP was obviously observed in hippocampus. PTEN mRNA and protein in hippocampus were significantly increased at VD group as compared to the sham group(p<0.01). PTEN expression were reduced apparently in hippocampus after RNAi intervention (p<0.01), however, no significant difference were observed in the AdRFP negative group (p>0.05).3. From the morris water maze results, there was obvious difference in each group. The escape latencies in VD model group were statistically longer, while the number of crossing the former site of the removed hidden platform were significantly less than those in sham group (p<0.01); As dealt with adenovirus siPTEN, the ability of learning changed in VD rats. Compared with the AdRFP negative control group, the escape latencies in siPTEN group were statistically reduced while the numbers of crossing the platform were significantly increased(p<0.01); The results of histopathological examined by Nissl staining and HE staining showed that neuronal damage of hippocampus in siPTEN group was much lighter than that of VD model and AdRFP group.4. CREB mRNA and protein in siPTEN treatment group were both significantly higher than those in AdRFP as well as VD model group(p﹤0.01), suggesting that the improvement of learning and memory in siPTEN group may be related to the increased CREB expression in hippocampus.Conclusion:1. In this experiment, with the use of 293 cells, we successfully amplified a large number of adenovirus AdR-siPTEN to meet the follow-up test needs.2. Not only adenovirus transfect ischemic dementiahippocampus tissue efficiently, but also PTEN-specific shRNA gene transfer conduct effectively. And the adenovirus-mediated RNAi effectively and stably inhibit the expression of PTEN in hippocampus.3. Vascular dementia rats have deficiency in the ability of spatial learning and memory. Adenovirus-mediated RNAi down-regulated PTEN gene can significantly reduce the damage of hippocampus neurons as well as the hippocampal degeneration caused by ischemic dementia, increase the expression of CREB in order to improve the ability of spatial learning and memory in vascular dementia rats efficiently.