| Objective：Source conventional cellular immunotherapy of cells mostcome from leukemia in remission of leukemia patients，a certain percentage ofleukemia cells are present in the peripheral blood of relieved or untreatedleukemia patients，can stimulate anti-tumor CIK cells deserves to be furtherexplored. Peripheral blood were collected from remission，non-remissionacute leukemia patients，mononuclear cells were extracted and cultured asdendritic cells(DC)and cytokine-induced killer cells(CIK), DC and CIK cellproliferation were observed, immune film marked change and the effects ofleukemia cells were detected. The biological activity and anti-leukemia effectof Co-culture DC and CIK cells were compared between remission patientsgroups and non-remission patients groups.So we can looking for better waysto get effector cells for cell immunotherapy.Methods：DC cell cultivation:fresh RPMll640cultures were added to thehomologous adherent cells appealed to, which were blown and suckedrepeatedly that adherent cells become suspension, centrifuge and wash, countcells, adjust the cell concentration to1×106per ml, then were cultured usingRPMI1640culture medium containing10%fetal calf serum,recombinanthuman granulocyte macrophage colony stimulating factor (rh-granulocyte-macroph-age. Colony-stimulating. Factor, rhGM-CSF)1000U/ml, humanrecombinant interleukin4(rh-interleukin-4, rhIL-4),1000U/m wereadded. According to the conventional cultivation, medium is half changedevery two days,with cytokines added,then count the cells. On the sixth day ofDC cell culturation, recombinant human tumor necrosis factor-a (rh-tumornecrosis factor-a, rhTNF-a)1000u/ml was added, to induce the DC tomaturation.Culture CIK cells in vitro, Peripheral blood were collected from remission,non-remission acute leukemia patients,after heparin anti-coagulation,then the mononuclear cells were isolated by H-F (Hypaque-Fieoll)lymphocyte separation medium（relative density of1.077±0.001), washedthree times by RPMI1640broth，adjusting cell concentration to4×106per ml,after four hours’incubation, suspension cells were collected，and adjust cellconcentration to1×106per ml with RPMI1640medium which containing10%fetal calf serum，then cultured at37℃,5%CO2，join different cytokineson day1and day2，the replacement of the new culture medium with rhIL-2added were taken every two days，the cells were counted at the same time..Onthe seventh day,CIK cells and DC cells were colected,and living cells werecounted trypan blue exclusion method, adjusting the DC cell concentrationto1×106per ml and CIK cells to5×106per ml in RPMI1640liquidcontaining10%FBS. The two kinds cells were mixed to culture, according tothe ratio of the DC and CIK cells for l:5. On the first day,9th day，12th dayand15th day after co-culturation, the mixture were collected, counted, anddetermined the biological activity.Results：1Comparison of cell proliferation of the two DC-CIK groupsAfter incubation the adherent mononuclear cells by GM-CSF andIL-4-induced cells into DC, the training process significant changes in cellmorphology, cell volume also increased significantly. The first two days inculture the cells become larger, the surface cytoplasmic projections, irregularmorphology, with time culture, cell surface protrusions increased, longer, andgrow in clusters into the group(see Figure1).Non-adherent mononuclear cells induced by stimulation of variouscytokines, a part of lymphoid cells was amplified into CIK cells and graduallyform a cell pellet; the cell body was smaller than DC, initially circular,transparent, more uniform size(See Figure2).The first four days in culture, thecells began to show an irregular shape, and showed clusters growth(see Figure3).From the culture from the first six days, the cells proliferatedsignificantly,clumps or colonies growing state,and suspended in culture medium(see Figure4).As the incubation time, the cells clump and cellcolonies increased.In the first six days of cell culture to co-culture DC and CIK cells intoDC-CIK cells, different groups were compared in their changes of immunephenotype and biological activity. The first nine days of cell culture by trypanblue exclusion cell counting, multiple CIK cells proliferation was(15.46±1.60),the proliferation of multiple DC-CIK cells of remission patientswas(16.39±1.89)，and the DC-CIK cells of non-remission patients was(17.0±2.16).The first12days, the total number of CIK cell expansion to theoriginal (35.9±4.2)-fold, DC-CIK cells was of remission patients(42.6±3.93)times the original，while the DC-CIK cells of non-remission patients was (40.2±2.97).The first15days, the total number of CIK cell expansion to theoriginal(47.3±3.4)-fold; total DC-CIK cells of remission patients to theoriginal(65.2±1.81)-fold，and DC-CIK cells of non-remission patients was(65.4±2.56). Proliferation of multiple comparative analysis of the same groupof cells increases over time were statistically significant.(See Table1)that CIKgroup and the group of DC-CIK cells were proliferating evolve over time,thetwo.DC-CIK cells and CIK cells were morphologically similar microscope,showing DC and CIK cells aggregate into larger groups(see Figure5),showing clumps grow.2Molecular marker phenotype Changes and comparison between theDC-CIK and CIK cells.Cells were collected on the first day,9th day,12th day and15th day tocompare Molecular marker phenotype. The results showed that: DC-CIK cellsof remission patients cultured for first day,9th day，12th day and15th,theimmune phenotype test results were as follows: CD3+CD8+cells were (23.4±5.36)%,(53.0±1.58)%,(71.2±2.59)%,(87.5±1.29)%, CD3+CD56+cellswere (3.81±1.35)%,(31.4±1.14)%,(49.0±2.58)%,(63.8±1.98)%,andDC-CIK cells of non-remission patients’ results were (21.8±7.32)%,(54.0±2.98%),(71.4±1.82)%，(88.6±3.4)%，(4.01±1.63)%,(32.4±2.24)%,(50.0±3.2)%,(66.8±4.1)%.(See Table2) shows that DC-CIK cells of CD3+CD8+and CD3+CD56+were significantly increased, but the differentsbetween the two DC-CIK groups were not significant(p＞0.05).3Comparision of the froup of DC-CIK cells in cell killing activity toK562.Cells were collected on the15th day,experiments effector to target ratioswere5:1,10:1,20:1,DC-CIK of remission patients killing activity againstK562cells was（21.7±4.64）%,（28.9±3.79）%,（42.64±3.25）%，andDC-CIK cells of non-remission patients’ results were（30.40±2.73）%（,42.38±3.20）%,（57.49±2.87）%. The results show that the two group of cells’killing activity against K562cells are effector to target ratio increases andenhanced, and the non-remission group was harder,but the differents weresignificant (p＞0.05).4The quanlity of leukemia cells in DC-CIK source of non-remissionchange after culturationOriginal cells in peripheral bloods of non-remission were counted bysmearing, with an average of (8.3±3.1)%before culturation,flow measuredCD34+cells accounted for (4.23±3.48)%;mixed culture for some time beforesmears observed under the microscope again,original cells were not seen,CD34+cells was reduced to an average of (0.1±0.05)%under flowcytometryto detect. Compare the results, the original cell was significantlyreduced after been cultured,the difference was statistically significant,P<0.05.5Comparation of specific cells killing activity of DC-CIK source fromnon-remission.DC-CIK cells source were collected from non-remission,as effector cellsto K562cells and mononuclear cells of patients,the cytotoxic activity of theeffector to target ratio within the range of5:1to20:1,when K562cell killingactivity results:(30.48±2.94)%,(42.66±3.46)%,(57.22±4.23)%,simultaneousdetection efficiency in the corresponding target ratio of cryopreservedmononuclear cells of patients with killing results was (35.23±3.43)%,(47.65±5.92)%,(67.29±7.95)%,(see Table4), after statistical analysis, the target has the same effect as a significant difference between the two conditions thanlethal.Conclusion:1Application without remission in patients with peripheral bloodmononuclear cells, can also develop a DC-CIK cells, compared with theremission group derived DC-CIK cell proliferative activity was no significantdifference.2Without remission and remission after co-culture, DC-CIK cells withCD3+CD8+and CD3+CD56+double-positive cell ratio was significantlyincreased, there was no significant difference.3Without remission and remission DC-CIK cells co-cultured, the killingactivity to K562were significantly improved, difference between the twogroup was not statistically significant.4DC-CIK cells specific killing activity enhanced sources ofnon-remission, and leukemia cells were not residual after cultured.. |
PDF Full Text Request