Studies Of Human Umbilical Cord Mesenchymal Stem Cells Intravenous Injective Transplantation Via Vena Caudalis In Rats With Ischemic

Objective:Human umbilical cord mesenchymal stem cells (hUMSCs) were cultured and amplified in vivro.Observe and identify the biological characteristics of hUMSCs. Exploring the mechanism and effect of hUMSCs transplantation in the treatment of ischemic stroke, to provide a a theoretical and experimental basis.Methods:Umbilical cord from full-term pregnancy and healthy neonate was washed by D-Hank’s, the vein and amnion were stripped off, cut Wharton’s jelly into small fragments of1mm3,then were planted in petri dish by explant method,or digested by collagenase I,hyaluronidase and dispase,planted in growth medium, contain DMEM/F12,20%fetal bovine serum (FBS) and2ng/mL epidermal growth factor (EGF). Then obsevre morphological changes of the primary culture cells. When adherent cells reached about90%confluence, they were detached and were passaged.Collected Passege5cells. Then detected cell surface phenotype by FACScalibur with mouse monoclonal fluorescein isothiocyanate (FITC),labeled antibodies, which includes CD29, CD44,CD34,语CD45,CD105,CD106,HLA-DR. FITC conjugated mouse IgG were used as controls.Select the left middle cerebral artery occlusion model.Adult male Sprague-Dawley (SD) rats weighing in250～280g were employed as the experimental animals.A nylon suture0.26mm in diameter,was advanced from the left common carotid artery into internal carotid artery,until it blocked the origin of the middle cerebral artery,then MCAO was induced.2hours later,extracted the nylon suture,animals were reanesthetized.24hours later,The neurological function of experimental rats were evaluated by modified neurological severity score(mNSS).And the rats awarding7-12points were randomly divided into groups as follows:(1)hUMSCs transplant roup,n=13, Transplanted of100cells;(2) Phosphate Buffered Saline(PBS) control group, n=13, Transplanted by PBS.HUMCSs labeled by CM-Dil were injected via vena caudalis,each rat received approximately3×106hUMSCs. The control group were injected with PBS in the same way. before MCAO induced and at1,7,14,21,28day after MCAO, A set of mNSS tests was performed.Finally, the rats were sacrificed, the brain tisue was removed and were made to successive sections,and inspected by fluorescence microscopy.SPSS was used to analyse the data.Results:A small amount of cells with fibroblastic morphology were found in5days after Wharton’s jelly pieces planted,small colonies could be found about7days later,then the cells expanded, and reached80%-90%confluence after12days.By digestion method,24hours after cultivation,Adherent cells with with fibroblastic morphology could be found.Then expanded,and reached80%-90%confluence about9days later,which present a radiating appearance.The primay cells were passaged at a ratio of1:1,then were passaged at a ratio of1:2or1:3according to the cell number, dozens of minutes later the cells rapidly adherented.2-3days later,cells cultures reached90%confluence again with whirling-like appearance.FACS shown that hUMSCs express CD29,CD44,CD105and not express CD34,CD45, CD106,HLA-DR(HLA-II).Between1-14days after hUMSCs were transplanted, compared to control animals, rats of hUMSCs transplant group were significant improvement on mNSS (p<0.05).Immunofluorescence staining confirmed that hUMSCs survived in Ischemic area.Conclusion:In our study, we found that umbilical cord was a rich source of MSCs, which could be easily isolated, cultured and passaged in vitro.Transplanted hUMSCs could survive,and improve functional recovery after MCAO in rats. This study demonstrated that human mesenchymal stem cells are rich contented in umbilical cord, and could be easily cultured,isolated in vitro. By caudal vein transplant ation,hUMSCs could survived in the brain of rat, and improve the neurological function of rat with MCAO.