| Neonatal hypoxic ischemic brain damage is one of the main reasons that cause children's deaths and disability of nervous system.In the recent years,severe HIBD is still lack of specific and effective treatment.but now,with the progress of stem cell's research, stem cell transplantation for the treatment of HIBD opened up a new way. Stem cell transplantation in recent years, the field of medicine and the whole field of life sciences research focus, because of their high degree of self-renewal ability and differentiation potential to become a multi-cell transplantation and tissue engineering in the preferred cell.Many studies have indicated that human umbilical cord blood(HUCB) is rich in hematopietie stem cell/hematopietic progenitor cell(HSC/HPC), Umbilical cord blood stem cells transplantation have been found to be successfully used in clinical therapy diseases of blood system,if we can found mesenchymal stem cell from the Umbilical cord blood is still a problem. In recent years, study shows that we can culture mesenchymal stem cell from Umbilical cord blood.And the nature in the bone marrow mesenchymal stem cell and the Umbilical cord blood stem cells is similar. They both have the ability of Multi-differentiation. Under certain environment,it can differentiate into neurons and glial cells. Umbilical cord blood MSCs transplantation therapy in hypoxic-ischemic brain injury and brain spinal cord injury in animals, the transplanted cells were observed in brain tissue of neurons and glial cell marker expression and nerve function recovery.However, it is found that the number of umbilical cord blood cells were insufficient and it has brought a number of difficulties for experimental research and clinical work, some research reported that two umbilical cord blood cells after mixed culture can have fast, large quantity to solve the insufficient number of umbilical cord blood.ObjectiveObserve whether we can obtain umbilical cord blood mesenchymal stem cells (MSCs)from healthy full-term newborn cord blood, Observe the proliferation of mixed and single umbilical cord mesenchymal stem cells.To assess the effect of human umbilical cord blood mesenchymal stem cells by mixed culture of intracerebral transplantation in the neonatal rats with hypoxic-ischemic brain damage (HIBD).MethodsUnder sterile conditions, to collect 20 healthy full-term neonatal cord blood by density gradient centrifugation to isolate mononuclear cells from cord blood and then by adherent cells to develop human umbilical cord blood MSCs. When the cells were in the 3rd generation,8 parts of the cultured umbilical cord blood mesenchymal stem cells (half) were taken out at random for group A; 8 parts of umbilical cord blood mesenchymal stem cells (half) for group B; and then mixed the remaining half of the cord blood mesenchymal stem cells from group A and B as group C. The cell density of the 3 groups was 1.0×106/ml. The expression of the cell surface antigens, CD86,CD34,HLA-DR,HLA-ABC of the 3rd generation of cord blood MSCs cultured in a mixed way was detected by flow cytometry. P3 generation of human umbilical cord blood MSCs was chosen as transplanted cells, which was vitro marked by 52-bromo-uracil BrdU.507-day-old SD rats chosen as HIBD models,3 ones died in the process. Randomly selected 5 ones to HE staining.The remaining 42 ones were divided into the mixed transplantation group (n=16), the single transplantation group (n=16) and the control group (n=10). On the third day after modeling, in stereotactic conditions, human umbilical cord blood MSCs were infused into the rats of transplantation group by way of the left cerebral cortex, and the same volume of PBS was infused into the control group by way of the same position. On the 7th day after cell transplatation, the brain of 6 cell-transplanted rats selected randomly were taken out after being killed, and then the brain tissue was used to observe the survival, migration and diferentiation of the transplanted cells in the brain by the immunohistochemistry method. On the 1st,7th,14th,21ts, and 28th day after transplantation, the 3 groups of rats' neurological function was evaluated by improved neurological damage score (mNSS) method.RetultsThe living cell counts showed that two different umbilical cord blood mesenchymal stem cells were mixed, in which cell proliferation was faster than single cord blood mesenchymal stem cells (p<0.05). HE staining after HIBD models:we can see a large number of nerve cells is diminished,and the cytoplasmic shrinkage, nuclear enrichment deeply stained, nucleolus disappeared, the cytoplasm loose, interstitial edema.Pyramidal cells decreased, cell gap increases, disordered, normal neuronal loss. On the 7th day after cell transplatation,The brain tissue of the rats in transplantation group was detected by immunohistochemistry, and it showed that the cells could survive in the brain and migrated around the point of transplantation, and BrdU-positive cells in mixed transplantation group were more than those in a single group (p<0.05). mNSS test results showed that on the 1st day, mNSS of the rats in transplantation group was lower than those in control group, but the difference was not significant (p>0.05); on the 7th,14th,21st and 28th, mNSS of the rats in mixed transplantation group was lower than the control group(p<0.05).on the 7th,14th,21st and 28th, mNSS of the rats in mixed transplantation group was lower than the single transplantation group, but the difference was significant (p<0.05).Conclusions1 It can be obtain MSCs from human umbilical cord blood of full-time healthy newborns.MSCs from human umbilical cord blood express remarkable MSCs-specific immunophenotype.2 The cell proliferation of mixed culture MSCs of human umbilical cord blood was faster than single culture MSCs.3 Mixed culture of human umbilical cord blood mesenchymal stem cells (MSCs) may possess a good clinical outcome in the neonatal rats with hypoxic-ischemic brain damage (HIBD).
|
PDF Full Text Request