| [Objective] To explore the changes in calcium channel signaling pathways in VaD（Vascular Dementia, VaD） rats by replicating rat model of vascular dementia (VaD)and conducting behavioral and calcium channel-related signaling pathway detectionson it, and to analyze the possible mechanisms of memory cognitive disorders causedby such changes. At the same time further preliminarily investigate the effects andmechanisms of transplantation of BMSCs（bone mesenchymal stem cells, BMSCs）on the improvement of cognitive function in VaD rats and on calcium channels aswell as calcium channel-related signaling pathways, and to provide applicable data forthe clinical application of BMSCs.[Methods]（1） BMSCs were isolated and cultured by adherence screening method, afterpurification of the fifth generation cells, surface antigen identification was performedon the cultured BMSCs by flow cytometry, BMSCs differentiation into neuronal cellswas induced by bFGF（basic fibroblast growth factorbfic，bFGF）and BHA（BetaHydroxy Acid，BHA）, neuroepithelial stem cell protein (Nestin) and NSE (Neuronspeeific enolase，NSE) expressions in the induced cells were detected and identifiedby immunohistochemistry.24h before the transplantation of induced BMSCs, markerBrdU(5-Bromo-2-deoxyUridine，BrdU) with a concentration of10umol/L was addedto the BMSCs medium, and the cells were cultured for an additional48h, the labeledBMSCs were collected by centrifugation and set aside.（2）. Before modelestablishment, SD rats were subjected to the water maze test for screening of intelligence. The rats were randomized into the control group and model controlgroup, the modified Pulsinelli’s four-vessel occlusion method was adopted in themodel group to replicate VaD model. In the control group, only the separation ofvertebral and carotid arteries was performed, and the coagulation of vertebral arteriesand occlusion of carotid arteries were not performed.30days later, rats with cognitivedysfunction were screened from the model group and divided into the model controlgroup and the stem cell treatment group.10μL (approximately1×106cells/microliter)of post-induced BrdU-labeled BMSCs suspension was injected into the lateralventricle of rats by stereotaxic instrument in the stem cell treatment group, while thecontrol group and the medium injection group were injected with the same volume ofculture medium.30d after cell transplantation, water maze behavioral test wasperformed, then rats in each group were decapitated and brains were removed toprepare paraffin sections, and pathological changes in hippocampus of rats wereobserved by hematoxylin-eosin HE（Hematoxylin-eosin，HE）staining.（3）Theexpression of BrdU-labeled post-induced BMSCs in rat hippocampus was detected byimmunohistochemical staining. NOS enzymes and their types and NO were detectedin plasma and hippocampal tissue of rats, Hemocytes and total hippocampal RNAwere extracted by Trizol one-step method, the mRNA expression levels of CaM,CaMKII and NOS were determined by real-time PCR. Total hippocampal protein wasextracted, quantified by BCA assay, and the expressions ofcalcium/calmodulin-dependent protein kinase II (CaMKII) and calciumchannel-related NOS proteases were detected by Western Blotting assay. All indexdifference were compared among the control group, model control group and stemcell treatment group, and the differences in the content among each group wereanalyzed statistically.[Results](1) BMSCs were isolated and cultured by adherence screening method, afteramplification by passage in vitro, the flow cytometry results showed that the positiverate of expression of adhesion molecules CD90in fifth generation BMSCs was99.4%,positive rate of CD29was96.8%, positive rate of CD44was99.6%, and positive rate of CD45was3.7%. BMSCs can express Nestin and NSE after induced.(2) Comparedwith the control group, the mean escape latency was significantly longer in the modelcontrol group and the stem cell treatment group, after the removal of platform, thetime spent in crossing the platform for the first time was extended and the number ofplatform crossings was significantly reduced (P<0.05) in the model control group andthe stem cell treatment group. Stem cell treatment group had shorter escape latency(P<0.05), shortened time spent in crossing the platform for the first time, andincreased number of platform crossings compared with the model control group, andthe differences were statistically significant.(3)30days after transplantation, BrdUstain positive cell distribution was observed in the bilateral hemispheric brain sectionsof rats in the stem cell treatment group, BrdU positive reactants were brown,granularly or diffusely distributed in the nucleus.(4) Differences in the expression ofcalcium channel and calcium signaling-related indices among the control group,model control group and stem cell treatment group were compared and analyzed. Theresults showed that the activity, mRNA and protein expression levels of aboveenzymes in the model control group were higher than the control group (P<0.05),compared with the model control group, CaMKII, CaM, NOS and NO expressionlowered in the stem cell treatment group (P<0.05).[Conclusion](1) BMSCs can induced to neuron-like cell by bFGF/BHA vitro,Also BMSCs can express Nestin and NSE after induced.(2) The NOS and CaMKIIsignal expression increased mainly related calcium channel at VaD model rats，indicating that calcium overload were all involved in the pathogenesis of VaD.(3)BMSCs can be labeled by BrdU, and that BrdU-labeled BMSCs can survive in the ratbrain.(4) The lateral ventricular transplantation-induced BMSCs can improve thecognitive ability of VaD rats to some extent by lowering some enzyme activitiescaused by calcium overload. |
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