The Study About The Change Of Phenotype And The Biologic Activity Of Effector Cells Which In Vitro Amplified From Human Peripheral Blood Lymphocyte

Objective: To study a new method of in vitro amplification of lymphocyte from human peripheral blood mononuclear cells(PBMCs).Methods: In GMP laboratory system, 35 malignant tumor patients' PBMCs were cultured in OKM-100 and OKM-200 medium and induced and amplified by stimulating factor OKM-24. After amplifying, analysed the cycle and multiple of amplifying, counted and compared the number of cells before and after amplifying, and observed the survival rate of the effector cells. At the same time observed the adverse reactions of the patients after the infusion.ResμLts: Effector cells were found proliferated from the second day to the end; the cycle of amplifying was 13.46±1.20d; before the amplifying, the number of lymphocyte cells was 6.60±2.23×10~6, after amplifying the number was increased to 2.99±0.65×10~9, the multiple of amplification was 488.06±191.25; survival rate detected by Trypan Blue staining was 97.71±1.07 %; the examination of endotoxin, bacterium, fungus, mycoplasma and adventitious viruses were all negative. There were no obvious adverse reactions after patients got effector cells infusion.Conclusion: The in vitro induction and expansion efficiency of sensitized lymphocyte cells from malignant tumor patients peripheral blood under this culture system is pretty high, the operation is simple, and the effectors cells after amplification are of good biologic security. Objective: To study the changes of lymphocyte phenotype which in vitro amplified from 35 malignant tumor patients' peripheral blood.Methods: Inducing and amplifying the lymphocyte cells and mononuclear cells from peripheral blood of malignant tumor patients, using the flow cytometry to detect the percentage of CD3~+,CD19~+,CD28~+,CD25~+,CD29~+,CD45RA~+,CD45RO~+,HLA-DR~+,CD3~+CD4~+,CD3~+CD8~+,CD3~-CD16~-CD56~+,CD3~-CD16~-CD56~+,CD3~-CD16~+CD56~+,CD3~+CD16~-CD56~+,CD3~+CD16~-CD56~+,CD3~+CD16~+CD56~+,CD4~+CD25~+,CD4~+CD29~+,CD8~+CD28~+,CD8~+CD28~-,CD4~+CD45RA~+,CD8~+CD45RA~+,CD4~+CD45RO~+,CD8~+CD45RO~+,CD3~+HLA-DR~+,CD3~+HLA-DR~-,CD4~+HLA-DR~+,CD4~+HLA-DR~-,CD4~+HLA-DR~+,CD4~+HLA-DR~- cells in effector cells, and then comparing their percentage before the in vitro amplification.ResμLts: After amplification, the percentage of CD3~+, CD3~+CD8~+, CIK (cytokine-induced killer cells) and it' s subgroups CD3~+CD16~-CD56~+, CD3~+CD16~+CD56~-, CD3~+CD16~+CD56~+, CD45RO~+ and it's subgroup CD8~+CD45RO~+, CD8~+CD28~-, CD25~+, CD29~+, CD3~+HLA-DR~-, CD8~+HLA-DR~+ and CD8~+HLA-DR~- cells increase greatly(P<0.01); the percentage of CD19~+, CD3~+CD4~+, NK(natural killer cells) and it' s subgroups CD3~-CD16~-CD56~+, CD3~-CD16~+CD56~-, CD3~-CD16~+CD56~+, CD45RA~+ and it's subgroups CD4~+CD45RA~+, CD8~+CD45RA~+, CD4~+CD45RO~+, CD28~+ and it's subgroup CD8~+CD28~+, CD4~+CD25~+, CD4~+CD29~+, CD4~+HLA-DR~+ and CD4~+HLA-DR~-cells drop obviously(P<0.01); and there are no statistical significance on the change of the percentage of HLA-DR~+ cells and CD3~+HLA-DR~+ cells(the value of P was 0.137 and 0.423, (?)espectively).Conclusion: After amplification, the main parts of the effector cells wereCD3~+ T cells; the percentage of CD4~+ T cells decreased greatly while percentage of CD8~+ T cells increased obviously; the percentage of regulatory T cells (Treg) and CD19~+B cells were very small; there were no obvious difference in the percentage of activity T cells before and after amplification. Among enhanced CD8~+T cells, effective T cells (T_E) did not increase greatly, while memory T cells (T_M) enhanced significantly. The percentage of NK cells decreased, while the percentage of CIK cells increased obviously. After analyzing the phenotypes of the effector cells, in theory we knew that the effector cells no only have the cytotoxic activity that can directly kill the tumor cells but also have the capability to become the effective cells when infuse back to patients and stimulate by some kinds of antigents.Objective:①To study the change of the survival rate of effector cells which in vitro amplified from human peripheral blood lymphocyte;②To study the killing tumor cells activity of in vitro amplified effector cells.Methods:①Used the method that said in the first part of this subject to in vitro amplify 3 malignant tumor patients' peripheral blood lymphocyte, harvested the cells and caculated the concentration of the effector cells. Then conserved the effector cells in 4℃and 25℃condition, using propidium iodide (PI) to dye the effector cells and using flow cytometer to detect the survival rate of effector cells in this two different conditions in different time.②Used the method that said in the first part of this subject to in vitro amplify 3 malignant tumor patients' peripheral blood lymphocyte, and used the method that said in the second part of this subject to detect the percentage of NK cells and CIK cells in amplified cells, and then detected the cytotoxic activity of effector cells by MTT method.ResμLts:①The incipient survival rate of effector cells of patient 1 was 98.8% and concentration of effector cells was 7.4×10~7/mL. In 4℃condition the survival rate of effector cells dropped greatly at 102hs, while in 25℃condition we saw the obvious decrease at 48hs. The incipient survival rate of effector cells of patient 2 was 95.5% and concentration of effector cells was 5.8×10~7/mL. In 4℃condition the survival rate of effector cells dropped greatly at 60hs, and in 25℃condition it obviously decreased at 24hs. The incipient survival rate of effector cells of patient 3 was 97.1% and concentration of effector cells was 10.4×10~7/mL. The survival rate of effector cells dropped greatly at 84hs and 24hs in 4℃condition and 25℃condition separately.②The percentage of NK cells and CIK cells in patient 1's effector cells was 2.4% and 53.9%, and the killing tumor cells rate of effector cells in 12:1, 25:1 and 50:1 effector cells/target cells ratio was 64.9%, 70.8% and 83.4% separately. The percentage of NKcells and CIK cells in patient 2's effector cells was 0.6% and 69.5%, and the killing tumor cells rate of effector cells in 12:1, 25:1 and 50:1 effector cells/target cells ratio was 71.6%, 75.1% and 88.6% separately. The percentage of NK cells and CIK cells in patient 3's effector cells was 11.6% and 41.7%, and the killing tumor cells rate of effector cells in 12:1, 25:1 and 50:1 effector cells/target cells ratio was 62.9%, 67.3% and 74.8% separately.Conclusion:①The effector cells should be conserved in 4℃condition rather than in 25℃condition. If the incipient survival rate of the effector cells is lower and the concentration of cells is thicker, they survival rate will decrease quicker and greater.②The effector cells have strong cytotoxic activity of killing tumor cells, and this kind of activity is positively related to the percentage of CIK cells in effector cells.