Guanylate-binding Protein 2 Regulates The Maturation Of Mouse Dendrtic Cells Induced By ?-glucan

Due to the seriously environmental pollution,aging population,working pressure,and bad life style,the incidence of cancer is increasing year by year.Therefore,exploring the pathogenesis of malignant tumors and finding new and effective treatments are hot but difficult spots of medicine.In terms of current tumor therapy,the most classic method is still surgical operation combined with chemo-or radio-therapy.However,for some of the advanced,metastatic or,recurrent tumor,these traditional treatments have little effects,but bring the considerable side effects of radiation and chemotherapy,which can seriously damage the patient's immune system or accelerate the process of tumor progression.Cell biology and immunology researchers pay attention to human immune system,and put forward the concept of biological therapy and immune therapy.Through strengthening the function of the immune system,we hope to eliminate tumor cells,re-active the tumor-immune cycle,restore the killing effect of immune cells to tumor cells,control the progression of cancer and cure tumor fundamentally.At present,through the immune cell therapy,therapeutic antibodies,cancer vaccines,as well as a variety of means such as immune adjuvant,we can enhance the ability of immune cell to recognize tumor associated antigens and augment the body's ability to kill tumor cel s.Dendritic cells?DC?mainly originate from the bone marrow CD34⁺hematopoietic progenitor cell?CD34⁺HPC?,which are the most powerful antigen-presenting cells?APCs?discovered in immune system.DCs can effectively capture and processe the antigen,and then present the antigen-peptide complex to T cell in order to activative the immune response.DCs are regard as the center of immune response because of the role of bridge in innate immunity and adaptive immunity.Immunotherapy based on dendritic cells is utilizing this property to activate cytotoxic T lymphocytes and natural killer cells,making them the effective anti-tumor effector to eradicate tumor cells.Therefore,how to improve DC's ability on tumor antigen recognition and antigen presentation;how to promp DCs migrate to the lesion and how to maintain the vitality of DC and survival time are the key points of preparation a high effective of DC vaccine.Whole glucan particles?WGP?as an immune adjuvant,can effectively induce the maturation of antigen presenting cells?monocyte macrophages,dendritic cells?,increase the secretion of pro-inflammatory cytokines,induce the respose of innate and adaptive immune,and enhance the antitumor immune effect.Long non-coding RNAs?lncRNA?are non-protein coding transcripts longer than 200nucleotides.It can regulate the interaction between different gene regulatory elements?including miRNA,histones,transcription factors?to regulate various biological processes.As a new research hot,a lot of evidences show that LncRNAs play an important role in the epigenetic and transcriptional regulation of mammals.In the immune system,LncRNA also widely expressed,and actively participated in the development,maturation,differentiation and activation process of immune cells?dendritic cells,mononuclear macrophages,T cells,B cells,etc?.However,the mechanism of LncRNA in the immune system is not completely clear.In particular,little is known about LncRANs that regulate the biological functions of dendritic cells that need to further explore.By gene chip analysis,we compared the differences expression of genes and LncRNAs in two states of DC stimulated with or without WGP,screening a kind of mostly upregulation genes?guanosine binding protein 2,GBP2?and a kind of LncRNAs?Lnc-TCONS₀0021102,Lnc-T0?in mature DC.This study aimed to investigate the two kinds of molecules'influence on the maturation and function of DC.These data provide experimental basis and theoretical basis for the preparation of high efficiency DC vaccines and optimizing tumor immunotherapy.1.Guanosine binding protein 2?GBP2?regulates the maturation of DC cells induced by beta-glucan.Objective:to investigate the effect of GBP2 on the DC's maturation and the ability to promote the proliferation of T-cell in vitro.Methods:BMDCs were generated from mouse bone marrow cells in vitro by culturing in a medium in the presence of Flt3.On the third day cells were transfected by siRNA GBP2/Lipofectamine 2000,and WGP were added on the fifth day.After48 h of culture in 37°C,the harvested cells were used for further experiment.Real-time quantitative PCR and Western Blot were used to detect the transfection efficiency.ELISA was used to detecte the levels of IL-6,IL-10,IL-12 and TNF-a in the BMDCs.Flow cytometry was used to detect the expression of CD11c,CD80,and MHC-IIin BMDCs.T cell proliferation assay was used to test the ability of BMDCs for promoting T cell proliferation.Results:Comparingthe DCs cultured with or without WGP,we found thatthe expression of GBP2 was significantly higherin DCinduced by WGP.After silenced the expression of GBP2,the mature of DC was restrained,the BMDCs'surface markers includingCD11c,CD80,MHC-II were downregulated.The cytokines IL-6,IL-12 and TNF-a decreased obviously and the ability to promote the proliferation of T cells decreased significantly.Conclusion:the reduction of GBP2 could affect the maturity of DC cells induced by beta-glucan,and inhibit the function of T cell proliferation.2.Long noncoding RNA Lnc-T0 regulates the maturation of DC cells induced by beta-glucan.Objective:to investigate the effect of Lnc-T0 on the DCsMethods:BMDCs were generated from mouse bone marrow cells in vitro byculturing in a medium in the presence of Flt3-L.On the third day cells were transfected by Lnc-T0-shRNA/Lipofectamine 2000,and WGP were added on the fifth day.After48h of culture in 37°C,the harvested cells were used for further experiment.Real-time quantitative PCR was used to detect the transfection efficiency and the mRNAs expression of immune function-related cytokines,including IL-6?IL-10?IL-12?TNF-??CCR7?iNOS.ELISA was used to detecte the levels of IL-6,IL-10,IL-12 and TNF-?in the BMDCs'supernatant.Results:We found that Lnc-T0 was significantly higher expression in DCinduced by WGP.After the expression of Lnc-T0 was knock-down,The cytokines of DC showed significant changes in mRNA level and protein level:?1?onthe mRNA level,IL-10and CCR7 were significantly decreased but TNF-?was increased in the experimental group cells;?2?on the protein level,the expression of IL-10 was decreased while TNF-?was increased in the BMDCs'supernatant compared with the Negtive control.Conclusion:Lnc-T0 can regulate the production of DC cytokines and related chemokine receptors at the transcriptional level,and also regulate the production of DC immune-related cytokines at the protein leve.However,whether the other effects of Lnc-T0 on DC and its mechanism remain to be further explored.