Effects Of Human Umbilical Cord Blood Mesenchymal Stem Cells On Diabetic Vascular Dementia Rats

PART 1 To isolation,culture,identification and marking of mesenchymal stem cells from human umbilical cord blood.[Objective] To isolation,culture,identification and marking of mesenchymal stem cells from human umbilical cord blood.[Methods] 20 parts of human umbilical cord blood were collected under sterile conditions, each 100 ml, and mononuclear cells were obtained within 6 hours by Ficoll density gradient centrifugation.Mononuclear cells were seeded at 106/ml in conditioned medium, and put in the 37°C, 5% CO2 cell culture incubator. Immunophenotypes of the cells surface were analyzed by flow cytometry. CM-Di I was labeled human umbilical cord blood mesenchymal stem cells.[Resulst] 10 parts of human umbilical cord blood from 20 parts could isolate and culture HUCB-MSCs, the success rate was 50%.Mononuclear cells which were seeded in complete medium after three days began to have a small number of adherent cells.The adherent cells proliferated slowly at the begining, and the adherent cells were more visible after five days.When the adherent cells could reach 80% in about four weeks, passaged in 1:3 ratio. Cells’ morphology was gradually becomed fusiform. The 3th generation cultured cells which were identified by flow cytometry could stably expressed HUCB-MSCs labeled antigen CD44, but could not express surface markers CD34 of hematopoietic cells.[Conclusion] Mesenchymal stem cells.could be isolated and cultured from human umbilical cord blood.PART 2 Human cord blood mesenchymal stem cells could improve to the abilities of learning and me mory in diabetes rats of vascular dementia.[Objective] To establish a replicable model of diabetic rats with vascular dementia. Human cord blood mesenchymal stem cells could improve the abilities of learning and memory in diabetes rats of vascular dementia.[Methods] 40 rats were randomly selected in 50 healthy male Wistar rats, and were made diabetic models by intraperitoneal injection of streptozotocin(STZ).30 rats could become diabetic. Vascular dementia models were prepared in 2-VO after one week. 40 rats were divided into control group(SHAM), diabetes mellitus(DM) group, vascular dementia(DM + VD) group, human umbilical cord blood-derived mesenchymal stem cells(HUC B-MSCs) groups,and every group had 10 rats.Rats in the DM group were separated the bilateral common carotid arteries,but the bilateral common carotid arteries could not be ligated.Before the bilateral common carotid arteries were ligated, HUCB-MSCs 2×106 were injected into the common carotid artery in the HUCB-MSCs group. Before the bilateral common carotid arteries were ligated, saline was injected into the common carotid artery in the DM + VD group. We could do the Morris water maze to test the learning and memory of rats after four weeks of HUCB-MSCs transplantation.[Result]Compared with the SHAM group, the rats in others had significantly longer average escape latency(P <0.05); and compared with the DM + VD group, the rats in the HUCB-MSCs group had significantly shorter average escape latency(P <0.05). Compared with the SHAM group,the numbers of throughing the original platform quadrant were decreased in the remaining rats(P <0.05); and compared with the DM + VD group, the numbers of crossing the former platform quadrant were increased significantly in the rats of the HUC B-MSCs group(P <0.05).[Conclusion]HUC B-MSCs transplanted by the common carotid artery could improve the learning and memory of rats which were vascular dementia combined with diabetic mellitus.PART 3 Human umbilical cord blood mesenchymal stem cells could impact the Ch AT and neuronal apoptosis of the rats which had vascular de mentia combined with diabetic mellitus.[Objective]To research if human umbilical cord blood mesenchymal stem cells could impact the C h AT and neuronal apoptosis of the rats which had vascular dementia combined with diabetic mellitus.[Methods] 40 rats were randomly selected in 50 healthy male Wistar rats, and were made diabetic models by intraperitoneal injection of streptozotocin(STZ).30 rats could become diabetic mellitus. Vascular dementia models were prepared in 2-VO after one week. 40 rats were divided into control group(SHAM), diabetes mellitus(DM) group, vascular dementia(DM + VD) group, human umbilical cord blood-derived mesenchymal stem cells(HUC B-MSCs) groups,and every group had 10 rats.Rats in the DM group were separated the bilateral common carotid arteries,but the bilateral common carotid arteries could not be ligated.Before the bilateral common carotid arteries were ligated, HUCB-MSCs were injected into the common carotid artery in the HUCB-MSCs group. Before the bilateral common carotid arteries were ligated, saline was injected into the common carotid artery in the DM + VD group. After the Morris water maze, immunohistochemical staining was observed the change of choline acetyltransferase(Ch AT) in hippocampal neurons of rats. Hippocampal tissue in each group was tested the neuronal apoptosis in the flow cytometry. Could HUCB-MSCs which were labeled in CM-Di I cross the blood brain barrier and act in the brain tissue of rats?[Result]HUC B-MSCs which were labeled in CM-Di I could be found in the brain, it wold be proved that HUCB-MSCs could through the blood-brain barrier of rat. Compared with the SHAM group, the number of Ch AT-positive neurons in others was significantly lower(P <0.05); compared with the DM + VD group, the number of Ch AT-positive neurons in the HUCB-MSCs group was significantly increased(P <0.05). Hippocampal tissue in each group was tested the neuronal apoptos is in the flow cytometry. Compared with the SHAM group, neuronal apoptosis in others became increasing(P <0.05).; compared with the DM + VD group, neuronal apoptosis in the HUCB-MSCs group became reducing(P <0.05).[Conclusion]HUC B-MSCs could improve the ability of learning and memory by increasing the number of C h AT-positive neurons and reducing neuronal apoptosis in the hippocampus.[Summary] 1、Mesenchymal stem cells.could be isolated and cultured from human umbilical cord blood. 2、The model of vascular dementia combined with diabetic mellitus is a replicable model. HUCB-MSCs transplanted by the common carotid artery could improve the learning and memory of rats which were vascular dementia combined with diabetic mellitus. 3、HUCB-MSCs could improve the ability of learning and memory by increasing the number of C h AT-positive neurons and reducing neuronal apoptosis in the hippocampus.