| Objective:The human umbilical cord mesenchymal stem cells, hUMSCs, were transplanted into rat intracerebral hemorrhage model to evaluate the survival, migration and neuronal differentiation of transplanted cells in the brain tissue of rats with bleeding injury and explore the effects of hUMSCs transplantation on the recovery of neurological function in rat intracerebral hemorrhage model and the mechanism behind. This research could provide a theoretical basis and experimental basis for the clinical application of hUMSCs in the field of neuroscience.Method:The umbilical cord from healthy neonatal with full-term pregnancy cesarean section was collected under the sterile conditions. Wash away the remnants of blood within the blood vessels by D-Hank’s and get rid of artery and vein in the umbilical cord. The remaining tissue was cut into small pieces with size of1mm3,digested by0.2%collagenaseⅡ and cultured in the DMEM/F12medium containing20%FBS (fetal bovine serum FBS),2ng/ml epidermal cell growth factor (EGFR of growth factor, EGF),25mM L-glutamine (L-of glutamine, L-Glu) and100U/ml penicillin,100μg/ml streptomycin. Trace the Morphological changes of primary cultured cells. When these cells grew confluent, they were digested and passaged by adding0.25%trypsin-1mM EDTA.Collect the digested cells and take flow cytometry to analyze the cell phenotypes including PE-expression of CD11b, PE-the CD45, PE-of CD73, PE-of CD90, PE-of CD105, PE-HLA-DR(HLA-Ⅱ), FITC-CD19, and FITC-of CD34, taking FITC or PE-conjugated mouse IgG1as the isotype control.Adult male Sprague-Dawley(SD)rats (270-300g) were selected in this study. According to Bao Xin-min rat brain stereotaxic location atlas, the right side of skull of rats (AP=-0.5mm; ML=3.0mm) were drilled with a1mm-diameter-dental drill. Then with the reference of Anterior fontanelle,2ul collagenase VII aqueous solution were slowly injected into the head of right caudate nucleus (AP=-0.5mm, ML=3.0mm, DV=5.5mm)from micro-syringe fixed on the stereotaxic instrument to build the rat intracerebral hemorrhage model.24h later, the neurological behavior of experimental animals were evaluate by modified neurological severity score (mNSS). Rats scored between7and12points(moderate injury)were chosen and randomly divided into three groups (1) hUMSCs transplantation group, n=20;(2)Phosphate buffered Saline, PBS,control group, n=20;(3) sham group, n＝5.The rats were anesthetized again, with head fixed in a stereotactic shelves. hUMSCs, marked with1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchlorate, Dil, was slowly injected into the edge of the bleeding lesions region(AP=-0.5mm,ML=3.0mm,DV=3.5mm)of the hUMSCs group rats with10μL Hamilton syringe. Each rats received10μL, approximately2×102cells. Fibrin glue closed the pinhole and the bone hole. PBS control group were injected the same amount of PBS,instead of hUMSCs, by the same method.In the1st,7th,14th,21st,28th,35th day after cell transplantation, the experimental animals were evaluated all with mNSS. Rats were executed and whole brain was removed for continuous frozen sections. Tested the survival and migration of the Dil marked hUMSCs in the tissue of intracerebral hemorrhage and the expression of microtubule-associated protein2, MAP2,and glial fibrillary acidic protein, GFAP, by immunofluorescence staining. SPSS15.0was used in the analysis of experimental data.Results:In the second day of cell culture, a small amount of different patterns of adherent cells were observed, scattering in the dish. About1week, the adherent cells become colonies, and then fibroblast colonies were checked which growing radically. About2W, when the cells confluence almost reach 90%, cells were digested in the ratio of1:2. A few hours after the passage,it appears rapidly adherent.1W later, cell confluence reach90%to95%.The cells were tightly packed swir1-like structure in the view of low magnification microscope.Flow cytometry analysis showed that these cells uniformly high express the MSCs sign of CD73, CD90, CD105,without expressing hematopoietic marks CD34, CD45, neutrophil signs of CD11b, human B cell markers of CD19. Analysis of the tissue compatibility phenotype displayed hUMSCs did not express human leukocyte antigen of HLA-DR (HLA-Ⅱ).1d after cell transplantation, there is no significant difference (P>0.05)between the mNSS scores of the hUMSCs transplantation group and the PBS control group. However, in comparison with sham group, there is significant difference(P<0.05).7-35d after cell transplantation, mNss scores of both hUMSCs group and PBS control group decline in various degree, but the score of hUMSCs group is more obviously (p<0.05).Changes of rats in hUMSCs group are more improved in terms of muscle strength, reaction time, balance and reflection. Although,the mNss score of hUMSCs group decline, some indicators compared with the sham group, there is still a significant difference (p<0.05)Immunofluorescence staining proved that, after35d,there were a certain number of hUMSCs survial in host brain after transplantation. In addition to parts of cells aggregate in the injection area and needle tract,the vast majority of transplanted cells distributed around the bleeding injured area. Besides, a small amount of transplanted cells were founded in the contralateral brain in hUMSCs group. Under the fluorescence microscope, some hUMSCs migrate into the vascular structure along the blood vessels toward the direction of arrangement, surrounded like red tubular fluorescent structures. Immunofluorescence staining confirmed that part of the cells can express MAP2and GFAP. It is worth noticing:these transplanted cells expressed MAP2or GFAP have no the typical morphology of neurons or glial cells.Conclusion:This study demonstrated that MSCs is rich in human umbilical cord and can be isolated, cultured, passaged in vitro.After transplantation, MSCs also can survive, migrate, differentiate in host brain and promote ICH rats recovery of neurological function. |
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