Research On Human Ucb-mscs Isolation,culture And The Effect Of IDO Expression On Immune-Mediated Aplastic Anemia Mice

PartⅠStudy on humam umbilical cord blood mesenchymal stem cellsisolation,culture and biological characteristicsObjective: To isolate and culture umbilical cord blood mesenchymal stem cellsfrom human umbilical cord blood in vitro, and approach the biological speciality, suchas the culture conditions, expression of the surface markers and multi-directionaldifferentiation potentiality and so on.Methods:1.Mesenchymal stem cells were isolated from umbilical cord blood withthe lymphocyte isolation method and hydroxyethyl starch sedimentation+lymphocyteisolation method. Compared the two methods’ culture results.2.The morphology ofUCB-MSCs was observed under inverted phase contrast microscope. The growth andreproductive activity of the cells draw into cell-growth curves.3. Selected the thirdpassage, surface markers of UCB-MSCs were detected and certificated by flowcytometry. To approach whether UCB-MSCs had the characteristics of mesenchymalstem cells.4.Selected the steady state cells,and induced by osteogenic and adipogenicculture. Then, Alizarin red, Oil red O staining were used to identify the induced-cells.Results: Compared with the lymphocyte isolation method,the number ofmononuclear cells significantly increased by the hydroxyethyl starch sedimentation+lymphocyte isolation two-step method. UCB-MSCs were adherent fibroblast-like cells,and after3-4weeks of primary culture,became parallel or spiral-like. Immunophenotypeanalysis showed that MSCs were positive expression for adhesion molecules markerCD44and stem cell characteristic surface marker CD73,but failed to expresshematopoietic lineage marker CD34. Adipocyte and osteocyte differentiation inducingsuccessfully, showed UCB-MSCs had the multi-directional differentiation potentiality.Conclusion: Umbilical cord blood mesenchymal stem cells were successfullyisolated and cultured in vitro.The cells had the biological characteristics ofmesenchymal stem cells. PartⅡ Study on IDO expression of UCB-MSCs induced by IFN-γand the effectof IDO in UCB-MSCs on allogeneic lymphocyte proliferationObjective:To study the IDO expression of UCB-MSCs induced by IFN-γ atvarious concentration and explore the effect of IDO in UCB-MSCs on allogeneicT-lymphocyte proliferation.Methods:After the third passage,the steady state UCB-MSCs were induced byIFN-γ at various concentration (0、50、100、200、400U/ml) for24hours.The IDOmRNA and IDO protein from UCB-MSCs were extracted and analyzed respectively byRT-PCR and Western blot methods.UCB-MSCs in different concentrations(UCB-MSCs:lymphocytes=1:1,1:10,1:50,1:100) were treated by mitomycin C and then lymphocytesuspension were added. Lymphocytes proliferation was detected by MTT assays.Results: UCB-MSCs failed to express IDO,but the IDO mRNA expression andIDO protein expression were found under the induction of IFN-γ.The expressionamount and the stimulant concentration of IFN-γ were in a dose-dependentrelationship.When various concentration UCB-MSCs (UCB-MSCs:lymphocytes=1:1,1:10,1:50),lymphocytes proliferation decreased obviously compared with control group(p<0.05).With the concentration of UCB-MSCs diminishing, the inhibition ratio oflymphocytes proliferation decreased. When UCB-MSCs: lymphocytes=1:100,lymphocytes proliferation had no apparent change compared with control (p>0.05).Conclusion: UCB-MSCs induced by IFN-γ could express IDO and UCB-MSCsmay perform the immunosuppressive function by the IDO expression. Part Ⅲ Study on the effect of IDO expression in humam UCB-MSCson immune-mediated aplastic anemia miceObjective: To establish murine immune mediated aplastic anemia model to explorethe effect of IDO expression on immune mediated aplastic anemia mice and theImmunosuppression mechanism.Methods:5.5Gy60Co γ-ray irradiated SPF BALB/C(female,8~12weeks) micetransplanted with DBA/2(female,6~14weeks) thymus/lymph node cells acted asimmune mediated aplastic anemia model. The following groups were simultaneously setted: Normal control group, AA group, MSCs transplanted group and MSCs with IDOexpression transplanted group.The14th day after the model established, UCB-MSCswere infused through vena caudalis at the dose of1×103per gram.The changes ofperipheral blood, pathological features of bone marrow and the hematopoietic negativeregulatory factor INF-γ level in all groups should be observed.Results: About7~16days after the aplastic anemia model established, the mice inAA group became pale, fur scattered, loss of appetite and weight loss about22.58±15.74%and after14days the peripheral hemogram and bone marrowhaematogenesis of model mice decreased apparently. UCB-MSCs with IDO expressionwere infused through vena caudalis as a treatment.The28th day in MSCs transplantedgroup, both of the peripheral hemogram and bone marrow haematogenesis recoveredand had obvious changes compared with the model group. The therapeutic effect of theIDO expression in UCB-MSCs on aplastic anemia was Statistically significant and thehaematogenesis function of bone marrow improved. apparently.Conclusion: UCB MSCs with IDO expression reduced bone marrow failure ofaplastic anemia mice by intravenous infusion.