| Objectives:1. To establish the differentiation system from hematopoietic stem cells to NK cells, and to explore the effects of different combination of cytokines on the cellular immunophenotype, differentiation mechanism, cell proliferation, and cytolytic activity of these NK cells.2. To analyse the role and mechanism of rhIL-15in the differentiation of umbilical cord blood drived CD34+hematopoietic stem cells towards NK cells.Methods:1. After dilution, cord blood mononuclear cells (MNCs) were isolated by Ficoll-Paque density gradient centrifugation. Subsequently, cord blood CD34+hematopoietic stem cells were obtained by magnetic cell sorting CD34microbeads using the Miltenyi MiniMACS device according to the manufacturer’s instructions. The CD34+hematopoietic stem cells were collected, and the purity of CD34+hematopoietic stem cells was tested by flow cytometry.2. The purified CD34+hematopoietic stem cells from umbilical cord blood were seeded at1×105cells/ml in24-well plates. lml complete RPMI-1640medium (with10%head inactivated fetal bovine serum,10-6mol/L HDC) was anded in per well. According to different combination of cytokines, CD34+hematopoietic stem cells were divided into3groups:group A (SCF+FLT-3L+IL-2+IL-7+IL-15), group B (SCF+FLT-3L+IL-2+IL-7) and group C (SCF+FLT-3L+IL-7). After3weeks culture, group C was divided into three subgroups C1(IL-2),C2(IL-15),C3(IL-2+IL-15) for another2weeks culture by adding different cytokine. Each group was seeded three wells. The cytokine concentration was SCF20ng/ml, FLT-3L lOng/ml, IL-2500U/ml, IL-720ng/ml, and IL-15lOng/ml.24-well plates were incubated at37℃in5%CO2humidified atmosphere for5weeks. Half of the culture medium was replaced with fresh medium containing10%FBS and the same concentration of growth factors every3-4days.3. The cells number of group A, B, and C was counted weekly by using cell counting plate. In group A and B, the percentage of CD3-CD56+NK cells was analyzed weekly by flow cytometry. In group C, the percentage of CD34brightCD122+NK progenitor cells was analyzed. In group C1, C2, and C3, the percentage of NK cells was analyzed.4. After5weeks of culture, CCK-8assay were used to analyze the cytolytic activity of NK cells in group A, B, and cord blood activated NK cells. Cord blood mononuclear cells were stimulated by IL-2500U/ml and IL-15lOng/ml for two weeks, and cord blood activated NK cells could be got. The cytolytic activity of induced NK cells was compared with that of cord blood activated NK cells. The target cells were K562and Hela cells.5. Gene expression levels of group A, B induced NK cells, and CD34+hematopoietic stem cells were detected by using real-time PCR. The expression levels of tumor necrosis factor, granzyme B, perform, IFN-γ, bcl-2, STAT5A, and STAT5B were analyzed.Results:1. The percentage of CD34+hematopoietic stem cells in umbilical cord blood mononuclear cells was only2.46%. After MACS sorting, the percentage of CD34+hematopoietic stem cells was88.7%.(3-6)×105CD34+hematopoietic stem cells could be got from each of umbilical cord blood unit.2. The percentage of NK cells in group A and group B was61.59%and17.91% respectively based on the results of flow cytometry. Each CD34+hematopoietic stem cell could generate an average122and21NK cells in group A and group B. In group A and group B, CD56+NK cells were73.4%and57.5%co-expression of the NKG2D receptor. It showed that IL-15enhanced CD34+hematopoietic stem cells to develop into NK cells, and promoted cell proliferation. IL-15also increased NK cells expressing of NKG2D surface receptor. The percentage of NK progenitor cells in group C was23.1%after three weeks culture. The percentage of NK cells in group C1, C2, and C3was7.45%,14.14%, and16.83%respectively for another2weeks. It was indicated that the combination of SCF, FLT-3L, and IL-7could promote CD34+hematopoietic stem cells to develop into NK progenitor cells. These precursors were then responsive to IL-15and became maturation into mature NK cells.3. At E:T ratios of10:1, NK cells had the highest cytotoxic activities. In group A, the kill rate of K562and Hela cell was24.7%and19.5%; In group B, the kill rate of K562and Hela cell was10.7%and8.5%. It was showed that IL-15enhanced NK cell cytotoxicity.4. Among the groups analyzed by real time PCR, expression levels of four genes(tumor necrosis factor, granzyme B, perforin, and IFN-y) decreased in the order A>B>>D34+hematopoietic stem cells. It was showed that induced NK cells had capacity for cytokine production. IL-15promoted NK cells to product cytokine. The expression levels of bcl-2, STAT5A, STAT5B, bcl-2/bax=2.356in group A were higher than those of group B. It was indicated that IL-15maybe mediated its effects by JAK3-STAT5signal pathway in CD34+hematopoietic stem cells differentiation to NK cells, and up-regulated bcl-2expression level to inhibit cell apoptosis.Conclusions:1. CD34+hematopoietic stem cells drived from umbilical cord blood could differentiate into NK cells in vitro. The best combination of cytokines was SCF, FLT-3L, IL-2, IL-7, and IL-15.2. The NK cells derived from CD34+hematopoietic stem cells were CD56bright subset, with secretion of cytokines and cytolytic activity to tumor cells.3. The combined of SCF, FLT-3L, and IL-7could promote CD34+hematopoietic stem cells to differentiate into NK progenitor cells, and the cellular immunophenotype was CD34brightCD122+.4. IL-15stimulated CD34+hematopoietic stem cells commit to NK cells. IL-15enhanced non-MHC restricted cytotoxicity of NK cells. |
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