A Study On The Therapeutic Effect Of Intravenous Administration Of Human Umbilical Cord Blood Cells On Vascular Dementia Rats

Background and ObjectiveThe repair of the damaged of mammalian central neural system (CNS) is very limited. The injured neurons almost cannot regenerate. At present, more and more studies focus on cells transplantation for the therapy of brain and spinal cord diseases. Fetal neural stem cells transplantation is one of the most effective method. However, transplantation of these cells is plagued with logistical and ethical considerations. Bone marrow cells can proliferate and differentiate into neurons and glial cells and express nestin antigen under certain conditions. Transplantation of bone marrow cells can treat CNS diseases. Human umbilical cord blood has the advantage of weak immunogenicity, immature lympholeukocyte, and is easy to be collected and protected, et al. Human umbilical cord blood contains more immature stem/progenitor cells than bone marrow. It is shown that human umbilical cord blood may have more practical value than bone marrow. It is reported that some researchers transplantated human umbilical cord blood cells (HUCBCs) into animal models of CNS damage. They observed neurological and behavior function recovery of the animals. Vascular dementia (VaD) is the damage of cognitive function. Cerebral infarction, low brain blood flow and haemorrhage are the reasons of the damage. Presently, VaD is considered to be the second reason of dementia in aged people, next to Alzheimer disease(AD). The rate of VaD has been increasing, but an effective treating method has not been found yet. Therefore, in the present study the effect of HUCBCs on improving cognition and behavior function was observed in VaD rats.Method1. The health puerperants without infectious disease and normal delivery and healthnewborns were chosen. Human peripheral blood were used as the control group.Ten cord blood and peripheral blood samples were collected, and the HUCBCs and human peripheral blood cells(HUPBCs) were isolated by standard Ficoll-Hypaque technique.2. Immocytochemical stain was used for identification if HUCBCs and HUPBCs express nestin antigen. The ratio of nestin positive cells and the cells cycle were analyzed by flow cytometer(FCM).3. The rats were randomly divided into a model group, a treatment group and a control group. The three groups were observed at 2, 4 and 8 week, respectively.4. Modified Pulsinelli 4-vessel occlusion (4-VO) was adopted to make a rat model of VaD. Within 3h after operation, the treatment group were administered with HUCBCs 3xlO6 /500|j.L through trail-vein.5. Immunohistochemical staining was used to identify the cells derived from HUCBCs in the brain in the treatment group.6. A widespread nuclear terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling (TUNEL) was used to identify apoptotic cells in hippocampi of the rats.7. Serum samples of rats were taken at 2w, 4w and 8w after operation. Neuron specific enolase (NES) and S-100 protein were analyzed by enzyme-linked immunoadsordent assay (ELISA).8. Light microscope and transmission electron microscope were used to observe the difference in pathology of rats' brain tissues.Results1. The nestin antigen positive cells were found in HUCBCs by immocytochemical assessment, while it was not found in HUPBCs. Flow cytometry demonstrated that approximately 4% of HUCBCs expressed nestin antigen. In the cell cycle, cells ratio of S in HUCBCs were obviously higher than that of HUPBCs, but the cells of Gm in HUCBCs were markedly lower than that of HUPBCs. There was no obvious difference in cells of G2/M between the two kinds of cells.2. There was no marked difference between the preopration AAR ratio in the model and treatment groups and the control group. The AAR ratio of the model group hadsignificant drop compared with the control group. The ratio of the treatment group had obvious improvement compared with the model group.3. MAB-1281 positive cells were found in the brain tissue of rats in the treatment group by immunohistochemical assessment, suggesting that cells derived from HUCBCs could enter the brain through blood brain barrier (BBB).4. Few TUNEL positive cells were found in hippocampus in the control group. TUNEL positive cells in the model group were increased to the highest level at 2w, and decreased at 4w and 8w, but yet obviously more than those in the control group. At 2w, the apoptotic cells in the treatment group were found obviously more than those in the control group, but they had no marked difference at 4w and 8w between the treatment group and the control group .5. In the model group, the serum NSE level was significantly higher than that in the control group at 2w, which restored to control level at 4w and decreased markedly at 8w. It was no significantly different between the treatment group and the control group at each time point.6. The level of S-100 protein in serum was significantly higher in the model group than that in the control group at each time point. It was no significantly different between the treatment group and the control group at each time point.7. Light microscope and transmission electron microscope assessment showed that the brain tissue in the model group had the pathology changes of chronic ischemia and hypoxia, such as pyknosis, condensation of the cytoplasm, lysosomosis in cytoplasm, axonal swelling, degeneration and demyelination. These damage was lighten markedly in the treatment group compared to the model group, meanwhile, huge nucleolus cells were were found in the course of brain repair.Conclusion1. In the present study, we found HUCBCs expressed nestin antigen, supporting that cord blood cells can adopt a neuralfate. It will provide theroy foundation for VaD treatment with HUCBCs.2. Cells derived from HUCBCs can enter into the rats' brain through BBB and lived inbrain.3. TUNEL positive cells in hippocampi in the treatment group was significantly lower than that in the model group. ELISA identified that serum NSE and S-lOOprotein level had not significant difference between the treatment group and the control group. Intravenous administration HUCBCs can protect neurons and glial cells, and reduce apoptosis cells.4. Behavioral tests shows the AAR ratio in the treatment group was significant higher than that in the model group, suggesting that HUCBCs therapy can improve cognition function of VaD rats.