Establishment Of Natural Killer Cell Induced Expansion System And Study Of Cell Function In Vitro And Vivo

Objective:At present,various studies have shown that Natural killer(NK)cells can induce an antigen-independent immune response against malignant cells and play an important role in tumor immunity.However,NK cells are only a small part of the immune system,accounting for only about 10%of white blood cells.It is present in a small amount in the human body.After humans reach the age of 25,immunity decreases and the number of NK cells decreases,while tumor patients and tumor implanted patients have fewer NK cells and are less active.Therefore,how to increase the number of NK cells in the body or culture a sufficient amount of active NK cells in vitro becomes the key to activate autoimmune to defeat cancer.In recent years,due to the development of NK cell expansion methods,the application of NK cells in adoptive cell immunotherapy has become possible.Now NK cells can be expanded into billions of activated NK cells from only a few milliliters of peripheral blood.There are many studies on NK cell expansion methods in Japan and China,however,the expansion methods at home and abroad are different and cumbersome.Therefore,the acquisition of high-quality NK cells is still the key to limit its clinical application.It is a great challenge to provide a large number of NK cells with good production standards and functional activities for patients.The purpose of this study is to explore how to establish a stable method of inducing expansion of NK cells in vitro,perform quality control on the expanded NK cells,and verify the anti-tumor activity of NK cells in vitro and vivo.Obtain sufficient experimental data to provide important theoretical support for NK cells to serve the clinical application of autogenous cell therapy products,and to remove a major obstacle in the field of NK cell immunotherapy.Methods:1.Establishment of natural killer cell induced amplification systemArtificial antigen presenting cells(aAPC)CD137LCD86CD64mIL-15-K562 were used as feeder cells and peripheral blood mononuclear cells(PBMC)isolated from human blood were co-cultured according to a certain proportion.The required cytokines were added to the medium,and stimulation culture was performed for 7 days as a cycle.NK cells were recovered after two cycles of stimulation and expansion,and trypan blue staining was performed to calculate the cell survival rate.2,Identification and quality control of induced and expanded natural killer cellsFlow cytometry was performed on the expanded NK cells,the purity of the expanded NK cells was analyzed(Percentage of C45+CD3-CD56+cells),and the expression of representative surface markers of NK cells(CD94,CD 15 8,NKp46,NKp44,NKG2D)was detected;reversed-Reverse transcription Polymerase Chain Reaction(RT-PCR)was used to detect transcription factors and identify NK cell lineages.3.Functional verification of natural killer cells in vitroWestern blot was used to detect cytotoxicity and apoptosis-related proteins of NK cells and tumor cells/fibroblasts in the co-culture system.The cytokine released from the supernatant of co-culture was detected by ELISA,and the cell co-culture tumor-killing experiments verified the activity of freshly cultured NK cells and frozen NK cells,and comprehensively evaluated the anti-tumor activity of NK cells in vitro.4.Functional verification of natural killer cells in vivoA549 tumor cells were injected subcutaneously into the back of the neck of the nude mice.When the tumor grew to a certain volume,NK cells were injected into the tail vein to treat the tumor bearing mice.The changes of body weight and tumor size of the treated and non treated mice were observed,and the antitumor activity of NK cells in vivo was preliminarily evaluated.Results:1.Establishment of natural killer cell induced amplification system:After 14 days of culture in vitro,NK cell expansion can reach 1000-1600 times,and the culture system has good repeatability and strong stability.2.Identification and quality control of NK cells:Flow cytometry showed that the representative surface markers of NK cells were stably expressed,and the purity of NK cells could range from 10.28±2.0%of peripheral mononuclear lymphocytes(PBMCs)initially to 88.95±6.17%of total cells after 14 days of culture.RT-PCR detection of transcription factors showed that transcription factors of different types of NK cells were expressed.3.Functional verification of natural killer cells in vitro3.1 NK cells have a highly effective killing effect on lung adenocarcinoma cells SPC-A-1,gastric adenocarcinoma cells BGC-823,lung small cell lung cancer cells A549,lymphoma cells Raji and NAMALWA,and leukemia cells K562,and the higher the ET ratio s(NK cells:tumor cells),the more obvious the killing effect,but for normal human embryo lung fibroblasts Walvax and foreskin fibroblasts HFF,NK cells do not produce a killing effect.After cryopreservation,the killing effect of NK cells was significantly reduced,but the tumor killing activity of NK cells was hardly affected under hypoxic conditions.3.2 Detection of cytokines in the supernatant:Co-cultured supernatants of tumor cells and NK cells expressed more perforin,granzyme B and IFN-y than NK cells alone.The expression of perforin,granzyme B and IFN-y in the co-culture supernatant of tumor and normal fibroblasts did not increase significantly;There was no difference in the expression of TNF-?,IL-2 and IL-6 in NK cell supernatant and co-culture supernatant.3.3 Protein detection:NK cells stimulated by tumor cell expressed more Perforin,Granzyme B,and Interferon-?(IFN-?)than NK cells alone.Tumor cells killed by NK cells express more apoptosis-related proteins Bax,Bad,and Cleaved Caspase3 than those untreated.3.4 NK cell treatment of tumor bearing mice:A549 tumor bearing mice after NK cell treatment did not lose weight,the tumor body was significantly reduced compared with the untreated group.Conclusion1.Co-culture of antigen-presenting cells with specific co-stimulatory molecules as feeder cells and peripheral mononuclear lymphocytes in proportion and exogenously adding cytokines can stably achieve the expansion of NK cells.Method is stable and repeatable,and the induced expansion of NK cells has a rapid expansion rate and high expansion purity.2.Under normal and hypoxic conditions,NK cells have high-intensity antitumor activity on a variety of tumor cells,including lung adenocarcinoma cell SPC-A-1,gastric adenocarcinoma cell BGC-823,lymphoma cell Raji and Namalwa,leukemia cell K562.However,NK cells have no killing effect on HFF and Walvax.3.After improving the NK cell freezed method,significantly enhanced tumor killing activity of NK cells,but the tumor-killing activity is lower than that of NK cells that are not frozen.4.NK cells kill tumor cells directly by releasing perforin/granzyme B and secreting IFN-?.NK cells only release a small amount of cytokines that cause cytokine storm,such as tumor necrosis factor ?,IL-2 and IL-6.5.The tumor size of A549 tumor bearing mice treated with NK cells was significantly reduced,and the induced NK cells had good antitumor activity in vivo.