| Objective:Present study aimed to explore the differences between dendritic cells(DCs) derived from CD34+ hematopoietic stem cells(CD34-DC) of the cord blood and those derived from monocytes(Mo-DC) of the peripheral blood, in their cell morphologies, cell phenotypes, and the function of inducing antigen-specific CTLs, which could specifically induce tumor lyses, and explore different sources DC ultrastructural changes related to its antigen processing and antigen-presenting function of differences, in order to explore more powerful therapeutic DC vaccines.Methods:Monocytes were obtained from peripheral blood of healthy volunteers by using density gradient centrifugation, and the dendritic cells derived from monocytes(Mo-DC) were harvested by adherent assay. CD34+ hematopoietic stem cells were harvested from the cord blood of healthy full-term pregnant women, and purified by using magnetic bead separation, and expanded in GM-CSF/SCF medium for 8-10 days. DC progenitors were differentiated continuously in the GM-CSF/IL-4 medium, and CD34+ stem cell derived DCs(CD34-DC) were harvested every 3 days. Take d3, d5, d7 days immature DC(CD34-im DC, Mo-im DC) and d7 harvested CD34-m DC, Mo-m DC(DC vaccine) in the TEM compare their cell processes, nuclear size and endosomal vesicles quantity. Taken after different incubation time(d1, d3, d5, d7) CD34-DC and Mo-DC surface molecules detected by flow cytometry expression, some cells pulsed with FITC-OVA257-264 and continued to culture 24 hours, respectively, detecting the antigen-presenting ability by flow cytometry. Take culture fifth day DCs load CEA protein and tumor necrosis factor(TNF-α) was added. Preparation of the mature CD34-DC, Mo-DC(DC vaccine), in vitro induced cytotoxic T lymphocyte(CTL) generation, both vaccines induced the CTL were of high CEA-expressing tumor cell lines Ls174-T do killing assay, using MTS cytotoxicity assay.Results:1 Morphologic appearance of immature DC was observed by an optical inverted microscope at d0, d3, d7 and matured status.When at d0, both of them were round or oval shape, with tiny projections from the membrane. from d3 Mo-DC seemed to have longer projection with fusiformis shape, while CD34-DC shorter but thorny. At d7, Mo-DC had prominent projections, straight and thin with the end needle liked, but CD34-DC shorter, thicker and rough membrane. CD34-DC displayed dendritic formation earlier or faster than Mo-DC. After stimulated by cytokine and tumor antigen, they displayed the relative consistency in cell appearance. Antigen loaded DCs were floated with no dendrite, round-like shape, and the membrane was clear and smooth.2 Transmission electron micrograph of DCs generated from CD34+ and monocytes.Take d3, d5, d7 CD34-im DC, Mo-DC and d7 harvested CD34-m DC, Mo-m DC(DC vaccine) per 50 cells in the TEM compare their cell processes, nuclear size and endosomal vesicles quantity. Number of processes on the surface of Mo-DCs was 9.05±1.2, 8.23±0.8 and 6.43±0.9 respectively at d3, d5, d7. Correspondingly, the number was 12.86±1.2, 16.82±1.5 and 11.87±0.3 in CD34-DCs. Processes number of Mo-DCs was decreased gradually, however increased at the first 5 days but reduced its number at day 7 on CD34-DCs’ surface. Size of nucleus of CD34-DC was 6.54±0.4, 7.34±0.3 at day 3 and day 5 of culturing, significantly larger than Mo-DC 5.1±0.2, 5.2±0.4 respectively. In the tumor antigen-loaded DCs, the nucleus size was 7.81±0.3 of CD34-DC and 6.23±0.2 of Mo-DC. Number of endosomal vesicles was determined and results showed CD34-DC had more endosomal vesicles than Mo-DC at each time points(P <0.05).3 The surface molecular(CD80, CD83 and CD86) expression were represented by positive cell percentage detected by Flow cytometric.Mo-DC d1-d7 had extremely low expression of CD80, while CD83 and CD86 increased gradually. After pulsing with tumor lysate and maturation cytokine all the three markers shoot to a high level. By comparison, CD34-DC lacked CD80 expression at day 1 but its level elevated at day 3, 5 and 7, and the positive cell rate reached to approximately 80% when matured by tumor lysate and cytokine stimulation. There are significant differences statistically between CD80 and CD86 expression on CD34-DC and Mo-DC surfaces, however when in mature state all of the three moleculars had similar phenotype(P >0.05, P >0.05, P >0.05). CD34-m DC and Mo-m DC generated from CD34-DC and Mo-DC stimulated by antigen and cytokine can become mature.4 The antigen presenting ability of DCs at different culture stages were represented by positive rate by FCM after pulsing with OVA257–264 peptide at d1, d3, d5 and d7 which is FITC conjugated.Results showed that nearly 15.27%±1.47 of CD34-DC were positive while only 5.16%±1.00 of Mo-DC. The positive cell percentage raised on day3, day5 and day7 but CD34-DC increased more significant than Mo-DC(P <0.05). When cultured on day 7 more than 81.8%±1.5 cells of CD34-DC were detected by FCM whereas only 57.2%±1.4 of Mo-DC. CD34-DC has more antigen presenting ability than Mo-DC.5 DCs stimulate allogeneic T cell proliferation by MTS assay.In our study, take DC of both source were cocultured with lymphocytes for 4 days at 1:1 and OD value of each group were recorded. The additional proliferation percent of lymphocytes resulted 208±39% of CD34-DC and 156±48% of Mo-DC, compared to the lymphocytes without DC stimulation as controls. From the view of mean velue CD34-DC induced higher amount of additional lymphocytes over Mo-DC, but there are no significant differences(P >0.05).6 CEA load generated CD34-m DC and Mo-m DC vaccine were activated CTL killing efficiency comparison of Ls174-T tumor cells with high expression of CEA.CC-CTLs showed 63.3±1.52%, 80.6±2.51%,90±2.0% cytotoxicity at ratio of 10:1, 50:1 and 100:1. By contrast, CTLs induced by Mo-DC targeted Ls-174 T cells with lower cytotoxicity percent, 35±2.64%, 53.6±3.21%, 68.6±3.51% respectively. Both of them displayed an increased oncolytic efficacy and CC-CTLs showed stronger cytotoxicity than CM-CTLs(P <0.05).Conclusions:1 With GM-CSF / SCF medium can induce cord blood CD34 + hematopoietic stem cell expansion and generate DC precursor cells and cultured adherent growth d8 beginning, a group can be harvested every three days DC precursor cells with GM-CSF / IL-4-induced medium can further generate CD34-DC, no phenotypic differences between CD34-DC and Mo-DC.2 Umbilical cord blood-derived CD34-DC has a stronger antigen-presenting, promoting lymphocyte proliferation and the ability to induce activation of CTLs.3 By electron microscope, changes of the number of dendritic protrusions, nuclear size and the number of endosomal vesicles are to assess in DCs’ micro-mechanism.4 Umbilical cord blood-derived CD34-DC is expected to become the new ideal raw cells for DC vaccine. |
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