| Objective: We mimicked hemopoietic microenvironment and implanted the expanded in vitro and non-expanded umbilical cord blood stem cells (UCBSC) in order to investigate their implant ability,improve implant conditions,enhance efficiency of transplantation,realize hemopoietic reconstruction and immune reconstruction and provided theoretical evidence for application of transplantation of UCBST to adults.Methods: We adopted cell culture in vitro,,CFU-mix and identification,cytochemistry staining,flow cytometer(FCM) in the following experiments.①Umbilical cord blood mononuclear cells(UCBMNC) were isolated using density gradient centrifugation and expanded in culture system containing cytokines. The plate was precoated with bone marrow stromal cells .Extension-adhesion test was performed to study their implant ability.②UCBSC were divided into three groups to test their implant ability:Group A : expanded UCBSCGroup B : non-expanded UCBSCGroup C : mixed UCBSC (1/3A + 2/3B)③Mixed cells were implanted into different feeder cells groups in order to select better implant conditions: Group A : umbilical cord blood stromal cells(UCBSTC)Group B : normal human bone marrow stromal cells(hBMSC)Group C : bone marrow stromal cells from acute leukemia patients in complete remission(BMSC-CR)Group D :basic fibroblast growth factor(bFGF)-treated BMSC-CR (bFGF-treated BMSC-CR)Results:①CD34+ cells were 2.41±0.41% of MNC isolated from umbilical cord blood with1.064g/L Ficoll-hypaque.The isolated cells formed CFU-mix in vitro easily.CD44+ cells were 2.35±0.39% of MNC.②CD34+ cells of MNC were proliferated almost 28.6 folds after 14 days′ culture in the presence of SCF, IL-3, GM-CSF and the number of CD44+ cells increased too.③The inplant ratio of MNC into hBMSC was 15.1±2.1% in non-expanded cells group, 12.3±1.6% in expanded cells group, 25.3±3.8% in mixed cells group.There was significant difference between mixed cells group and the other two groups④The implant percentage of mixed cells into UCBSTC(Group A), hBMSC(Group B), BMSC-CR(Group C), bFGF-treated BMSC-CR(Group D) was 42.2±5.6%, 25.2±3.8%, 23.4±3.6% and 34.8±3.3% respectively.Group A was remarkably different compared with the other groups(P<0.05). The forming ratio of CFU-mix in Group A(67.4±8.3/2×105)was higher than the other groups(P<0.05).The forming ratio of CFU-mix in Group D(51.4±6.7/2×105) was higher than Group B and C(P<0.05).The implant ratio of MNC in Group D(34.8±2.3%) was also higher than Group B and C(P<0.05).There was no significant difference between Group B and Group C(P>0.05).Conclusions:①The isolation effects of UCBSC using different density gradient Ficoll-hypaque were different. The content of CD34+ of isolated MNC with 1.064g/L Ficoll-hypaque was higher and the isolated UCBSC had better proliferation ability.②One part of each share of umbilical cord blood was cryopreserved and the other was expanded. Then the two parts were mixedly implanted, which could solve the problems of the shortage of UCBSC and the absence of stem cells owing to cell differentiation after expansion.③The implant ratio of UCBSC could be improved in bFGF-treated bone marrow stromal microenvironment from acute leukemia patients in complete remission. |
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