Therapeutic Study Of Bone Mesenchymal Stem Cells On Rat Model Of Vascular Dementia

Part 1 Isolation,cultivation and identification of rat bone marrow-derived Mesenchymai Stem Cells(MSCs)[Objective]To study the isolation,purification of rat MSCs in vitro. Detecte the biological features of MSCs.Identify and label the MSCs in the study.[Methods]We selected suitable culture medium and FBS concentration, separated and purified the MSCs with adherent method.The morphology of MSCs was observed with invert-microscopy constantly.The ultrastructure of MSCs was observed by transmission electron microscope.MSCs'curve of growth was depicted.MSCs were induced to differentiate into osteoblasts and adipocytes in suitable induction medium.MSCs were marked in vitro by BrdU. Immunohistochemical stain and light microscope were used for identification BrdU marker ratio.[Results]MSCs could be isolated and proliferated by adherent method in vitro.The appearence of MSCs were fibroblast-like cells and its growth curve was like shape of S.Under the electron microscope MSCs had plentiful rough endoplasmic reticula.The ratio of the nucleus to cytoplasm was high,and the surface of MSCs had a lot of microvilli.MSCs could be differentiated into osteoblasts and adipocytes in vitro.The incubation with 10μmol/L BrdU for 48h achieved over 90%of the labeling rate.[Conclutions]MSCs can be isolated,pured and expanded easily by adherent method.MSCs have the characters of earlier immature cells.MSCs have mutipotency which can be induced to differentiate into osteoblasts and adipocytes.The optimal BrdU labeling concentration and time period are 10μmol/L and 48h.Part 2 Transplantation of MSCs improved cognition of VD rats and enhanced the expression of BDNF[Objective]To construct the model of VD rats.To observe of MSCs improve cognition of VD rats.To observe the pathology and the expression of BDNF in rats' brain tissues after transplantation of MSCs.[Methods]①The rats were randomly divided into a sham group(n＝10);a model group(n＝15),10μ1 PBS was injected into rats'hippocampus at 30 days after 2-VO;a treatment group(n＝15),1×10~6 MSCs was transplanted into rats' hippocampus at 30 days after 2-VO.②The performance of the rats in Morris water maze were tested at 4 weeks after transplantation.③Hematoxylin eosin staining was used to observe the pathology of rats' brain tissues.④Immunohistochemical staining was used to identify MSCs in VD rats'brains and analysesed the expression of BDNF in brain tissues of rats in all groups.[Results]①There was no marked difference in the sham group,model group and treatment group before 2-VO.4w after transplantation,compared with the sham group,the average escape latency of model group was longer(P＜0.01),and the lingering time in platform area was shorter(P＜0.01).But the performance of treatment group had obvious improvement compared with the model group.②Light microscope assessment showed that the brain tissue in the model group had the pathology changes such as pyknosis and condensation of the cytoplasm.These damage was lighten in the treatment group compared to the model group.③BrdU positive cells were found in the brain tissue of rats in the treatment group by immunohistochemical assessment.④4w after transplantation,the number of BDNF positive cells in model group was less than sham group(P＜0.05).But BDNF increased in the treatment group(P＜0.05)[Conclutions]①MSCs which was transplanted into VD rats' hippocampus can live and migrate in VD rats'cerebral,and improve cognition of VD rats at 4w after translpantation.②The transplanted MSCs could enhance the expression of the BDNF.It suggests that the improvement of cognition of VD rats is probably due to the increased BDNF.