Study On Isolation And Culture Of Mesenchymal Stem Cells From Human Umbilical Cord And Transplant In Treating Mouse Of Aplastic Anemia

Objective: To establish a method of the isolation and culture of umbilical cordmesenchymal stem cells from human umbilical cord in vitro, and explore its biologicalspeciality. Methods:1. Human UC-MSCs were isolated and expanded in culture withexplantation technique, enzyme digestion（collagenase Ⅱa nd trypsin）respectively; cellmorphology with Wright, After the third passage, their surface markers were detectedand certificated by flow cytometry.2. The third and tenth passage cell proliferation wasanalyzed by MTT assay, P3, P10cell growth cycles were observed by flow cytometry.The changes of two passages were compared in cell growth and proliferation.3. Selectstate of the more stable4th passage cells(2×104/ml)，All cells were seeded into six-poreplaque that was played sterile coverslip handled with poly-Lysine. When reached needconfluence, UC-MSCs were induced to adipocyte and osteocyte differentiation withcorresponding to the induction system. When lique drops formed in cytoplasm, cellswere dyed with0.375%oil red O. Three weeks late, differentiatial osteocytes were dyedwith alizarin red.4. RT-PCR was used to detect the mRNA expressions of SCF、G-CSF、GM-CSF、VEGF in UC-MSCs.5. After UC-MSCs were co-cultured with peripheralblood mononuclear cells, the impact on the T cells were observed by measuring theproliferation by MTT method and the change of UC-MSCs were observed by Hoechstand PI double staining. Results: At about one week after explantation, thefibroblast-like cells were sprawled out from the edge of the tissue, growing in cluster; at3-5days after enzyme digestion, cells were watched growing in scattered. Afterpassageed, UC-MSCs were showed the status of long, spindle-shaped and circinategrowth. They have large nucleus, clear nucleolus. Immunophenotype analysis showsthey were strongly positive expression for CD29,(95.71±2.23)%, but negativeexpression for CD31,CD34,(2.47±0.54)%and (3.24±0.34)%respectively; the tenthpassage UC-MSCs still have strong proliferation ability, there were no distinct changes between the third and the tenth passage cells; UC-MSCs can be induced to adipocyteand osteocyte differentiation. RT-PCR results showed that mRNA of SCF、G-CSF、GM-CSF、VEGF expressed in UC-MSCs. UC-MSCs can suppress T cells proliferationin dose-dependent manner, otherwise, lymphocytes don’t affect the growth ofUC-MSCs. Conclusion: Building a method of umbilical cord of mesenchymal stemcells separation successful; The cells isolated from the umbilical cord have biologicalcharacteristics of mesenchymal stem cells. Objective: this study was to explore the effects of transplantation with human umbilicalcord mesenchymal stem cells (UC-MSCs) on bone marrow hematopoietic function inthe mice of drug combination-ray-induced aplastic anemia (AA). Methods:6～7weeks,120female Kunming mice were selected, the model of aplastic anemia mice wasestablished by combining iP cyclophosphamide and chloramphenicol after theirradiation with60Co γ-ray reference to the methods of Sunjiyuan et al. The experientwere divided into normal control group, model group and MSC group. In MSC group,human UC-MSC1×106was infused into per kilogram mouse by intravenous injection at2days after the last administration at one time. Survival rate, changes of peripheralblood, the number of nuclear cells in bone marrow, pathological features of bonemarrow and the changes of hematopoietic stem cells of the mice were observed in allgroups. Results: In AA model group, mice were dying at day8and death day10, theorgans were found pale through autopsy, the survival rate lower than those in MSCgroup (p <0.05). The number of white blood cells, platelet, Reticulocyte in peripheralblood decreased, the number of red blood cells did not change significantly at day10.Pancytopenia was found in model group and MSC group at day16, the others exceptRBC were recovery wildly, but recovery in MSC group was significantly, the differencebetween two groups was statistically significant. During the establishment of model, thenumber of nuclear cells decreased obviously in mice bone marrow, adipose tissueproliferated later period. After transplanted UC-MSC, the level of gamma interferon hadbeen declining than before. The number of nuclear cells and granulocyte-monocytecolony forming units in MSC group bone marrow was significantly higher than those in AA model group but no change had been found in colony-forming unit fibroblast.Conclusion: UC-MSCs infusion reduces the degree of bone marrow failure, andimproves the survival rate of aplastic anemia model mice.