For By Mobilization Of Peripheral Blood Mononuclear Cells In Vitro Expansion And Activation Studies

Currently,allo-HSCT is one of the most effective methods to treat the malignant hematologic diseases.For the past ten years,NST has witnessed fast development owing to its advantages in terms of safety and effectiveness.NST principally has two parts including the pretreatment as well as the immunotherapy before and after transplantation,between which the latter is more important than the former.The immunotherapy,depending on the GVL effect induced by the donor's immune cells, can extend the patient's post-treatment paracme,eliminate the minimal residual disease of leukemia.So far,DLI,as the classical therapy in the post-transplantation cell immunotherapy,has been used most widely in clinical treatment.However, people have discovered some disadvantages of DLI gradually in clinical practices.For example,it may induce GVHD,bone marrow depression and insecondary aplastic anemia.To solve these problems,we carried out the study of DSI in recent years, finding that DSI has the clinical potential to replace DLI due to its better GVL effect and other advantages such as reducing the occurrence of GVHD and preventing the bone marrow depression.Along with the rapid development of allo-HSCT and adoptive cell immunotherapy,donor G-PBSC,as the major sources of transplantation and cell immunotherapy,has become more and more important in clinical treatment. Nevertheless,most of the studies about G-PBSC in the past focused on studying the hematopoietic stem cells in the G-PBSC.Accompanying the improvement of clinical treatment,people are attaching greater importance to the immunological effect of G-PBSC.Nowadays,the direct infusion after collection is the only way to utilize donor G-PBSC,and the immune regulation of G-PBSC has not been brought into play totally.Therefore,it is urgent to search for an activation strategy which can not only induce and promote the GVL effect of G-PBSC but ensure the safety in clinical treatment in vitro.In physical stimulation and chemical induction,we selected the low-intensity He-Ne laser and rhIL21 to conduct the following experiments.In the first part of our experiment,we firstly used the low-intensity He-Ne laser with different dose intensity to irradiate the donor's mobilized peripheral blood,then isolated G-PBMNC from the blood and cultivated it in several groups.By CCK-8 assay,we proved that the low-intensity He-Ne laser with dose intensity between 0.9J/cm~2 and 4.5J/cm~2 could promote the proliferation of the donor G-PBMNC.We also clarified the relation between dose intensity and effectiveness,and found out the suitable dose intensity.At the same time,we detected the cytotoxicity of G-PBMNC in vitro on K562 cells by flow cytotoxicity evaluation using CFDA-SE/PI.The results showed that the laser can enhance the cytotoxicity of G-PBMNC while changing its proliferation activity.We also obtained the dose intensity when the induced G-PBMNC had the strongest toxicity.Subsequently,we,by flow cytometry evaluation,detected the effect of laser on the T cell subgroups of the donor G-PBMNC which has been cultivated for 24-72 hours.The results showed that no obvious changes occurred in the various subgroups of T cells within 72 hours after laser irradiation.We also detected the ability of Th subgroup and Tc subgroup in T cells to secrete IFNγand IL4 by flow cytometry using four-color staining,and investigated the relative mechanism about the immunomodulatory effect of He-Ne laser on the G-PBMNC.We found that the laser had no obvious effect in inducing the secretion of IL4,but it could stimulate the secretion of IFNγ.We also found out the two dose intensity which could induce the secretion of IFNγmostly.Finally,we,by chromosome aberration test and micronuclei test,proved that the low-intensity He-Ne laser with dose intensity between 0.9J/cm and 4.5J/cm2 had no chromosome teratogenic effect on the donor G-PBMNC and it could be used safely among this range of dose intensity.It is well known that IL2 is effective in some cancer immunotherapy.However, its use appears paradoxical in view of its primary role in immune-regulation.For this reason,searching for alternative candidates for cancer immunotherapy is still a great challenge.In the second part of our experiment,we used IL21,the most recently discovered member of the IL2 cytokine family,and some relevant cytokine combinations to induce the donor G-PBMNC in vitro.By flow cytotoxicity evaluation using CFDA-SE/PI,we proved that rhIL21 can enhance the anti-tumor activity of donor G-PBMNC in vitro,and its induction effect is comparable to that of rhIL2.We further used rhIL21 as the substitute for rhIL2 to improve traditional CIK cell induction system and obtained ideal results,showing rhIL21's clinical potential to replace rhIL2.Through comparing the cytotoxic activities of different cytokine combinations,we found out one induction combination which had the strongest cytotoxicity,which offered a new strategy to enhance the donor G-PBMNC activation in vitro.The changes of T cell subgroup and NK cell subgroup of the G-PBMNC induced by cytokines in combination with/without IL-21 were analyzed by flow cytometry after 3 or 7 days of culture,we found that the induction combination mentioned above had a significant advantage in inducing CD3~+CD8~+,CD3~-CD56~+ and CD3~+CD56~+ cell subgroup,we also analyzed the secretion of effector molecules related to immune function,including Perforin,Granzyme B and IFN-γ,at different times by RT-PCR,and further clarified the mechanism of cytotoxicity of the activated G-PBMNC.Finally,comparing with the most-widely-used traditional CIK cell induction system by cytotoxicity and cell subgroup analysis,we evaluate the effect of the cytokine combination which we picked up.The results showed that the cytokine combination had advantage over the traditional CIK cells induction system in inducing CIK cells.