Theanine Regulates T Lyphocyte Through Dendritic Cells And Inhibits Human Lung Adenocarcinoma Cells In Mice With Severe Combined Immune Deficiency

Objective:Lung cancer has become the malignant tumor of highest incidence and death rate in the world.In recent years,morbidity of lung cancer and mortality have increased,every year over one million new patients appeard in the world.Lung cancer incidence in China has been an upward trend,in the past 30 years,has been the incidence of lung cancer up to 61.4/10.By 2015,our country will become the first lung cancer in the world.Although chemotherapy,radiation therapy,harrow to the treatment of molecular technologies have developed continuously, efficacy of lung cancer treatment is still poor in overall.Biological response modifier(BRM) has opened up a new field of biological treatment in Cancer,the current BRM is a wide range of great importance to the prevention and treatment program of Cancer,which is commonly thought high of by medical science realm.it is the regulation and recoverying the immune function of the body.The effect mechanism of BRM is regulating the immune system by activating the special regulatory immune function of the body,stimulating the body's natural immune function,activating host immune defense system,and enhancing or restoring the host immune response to tumors.Cancer cells can evade the host's immune surveillance system,so that the growth and metastasis can be conduced.In the course of the generation of the body immune custody function and tumor-specific immune response,DC is the important joint of immunosuppressive to cancer,cancer show inhibition effect on DC,it can be conduce immune tolerance to tumor cells,so that T lymphocyte is leaded to destruction and loss of targeting specificity.DCs are antigen presentation cells which have strong function.,they possessed the ability of activating resting-type T cell and inducing ability.So,DC has become one of the attention focus of nowadays tumour biotherapy sphere because of its biology speciality,people have been paying more attention to DCs' clinical application because of its impotant function on body of immunity especially on its anti-tumor effect.At present,initial achievement had showed about the application of DC's tumourtherapy,a series of animal experiment showed that DC could induce special effection of anti-tumor protection,and then tumour could be resolved and stabilized.But many problem must be researched and solved,such as:the technique of non-serum cultivation is still immature,many stimulating factor must been added to promote DC generating and ripening,but these factors must be identified;it also isn't identified that which method allergizing DC could provocate more effectively the immunoreaction of body anti-tumor;the best method of DC retro-transfusion and the quantity,path,frequency and interval time definition etal of inoculating DC clinical application are immature.So,looking for simple,economic and effective stimulator as immunoadjuvant is one direction of lung cancer therpy in future.With the investigative deepenness and the technic flawless,DCs have extensive clinical application prospect on the tumor precaution and therapy,they would be generally utilized clinically.Theanine can prevent and treat tumour through a variety of mechanisms,it could interfere in many joints of tumor occurrence on biochemistry and molecular level,including tumor initiation,promotion and development,regulating the immune organ function and preventing immune suppression,delaying tumor-bearing animals immune recession, enhancing immune function and enhancing tumor-specific and non-specific immunity,obviously enhancing the body anti-cancer"main force"of cytotoxic T lymphocyte killing tumor cells.Theanine is a type of safe and atoxic food.Above of that,Theanine has been confirmed safe material by Japan and USA,which isn't limited dosage regulation in use. Preliminary experiment use mimicry tumor cell excreting cell factor environment and use bombesin(lung cancer cell excrete) co-culture with DC,and it confirm bombesin could obviously decrease DC quantity when its' co-culture with DC pre-cell,and HLA-DR,CD86,CD83,CD80,CD40 express obviously lower in DC,the capability of DC stimulating leukomonocyte reaction descends,HLA-DR,CD80,CD86,CD83,CD40 expression obviously increase after Theanine stimulating DC,Theanine could strengthen the ability of stimulating leukomonocyte reaction,and they got close to normal control group.So,Theanine could inhibit DC apoptosis and increase DC function with bombesin,it can increase the ability of DC stimulating T cell proliferation,we presumpt Theanine might prevent the inhibition to DC in the tumor environment,and then study,if Theanine could prevent and cure cancer as food additive.Methods:Simulate the inhibition of DC caused by bombesin that secreted by tumor cells in tumor environment,set up a bombesin suppress DC model,with intervention of theanine,prove the protection and activation influence on secreting bombesin to inhibit DC in tumor environment caused by theanine through the influence on DC morphology and functional maturation,and the expression of COX-2 mRNA and protein.There are 3 groups:the bombesin+Theanine DC group; bombesin+DC group;PBS+DCgroup.Use bombesin to suppress DC,compare to the DC in normal group after the intervention with theanine,then observe the influence of different theanine's concentration on the inhibition of DC surface molecules and IL-12 caused by bombesin(dose-effect of growth curve),analysis of the inhibition of DC apoptosis,DC morphological inspection,as well as the influence on expression COX-2 mRNA and protein.In vivo experiment,inject human peripheral blood lymphocytes into SCID mice to rebuild the immune,then use theanine subjects factors to stimulate DC cells,use A549 line to inoculate under the SCID mice's skin,approve the immune protection and immune therapy influence of theanine on DC through observing the transplanted tumor SCID mice model.Cultivate A549 cells,isolate and cultivate DC according to the original method.There are theanine stimulating DC group,DC group,theanine group and PBS group,adjust DC to the same cell concentration of 6.25×10⁵/ml with homogeneity cultivate solution,add it to 24-hole culture plate with 2ml for each hole, cultivate in 37℃and 5%CO₂ for 7days to be reserve,while for theanine group taking the DC suspension cultivated by the above-mentioned method for 2 days,add the leaching of theanine to make the final concentration to be 200μmol/L(optimal stimulus concentration),cultivate according to the method mentioned above.In the SCID mouse tumorigenicity experiments,by intraperitoneal injection with DC, A549,DC,PBS and theanine separately,and then doing the histopathological examination by using Optical microscope to observe the tumorigenicity.Immune reconstitution in SCID mice experiments.To test the IgG levels in SCID mice by ELASE.The experiment to test inducing anti-tumor immune protective effects,observe the latency of tumor,size,weight,survival time and histopathological examination.The anti-tumor therapy experiments,observe the incubation period of tumor,tumor size,weight,survival time,parallel histopathological examination.Histopathological immunohistochemical detection,observe the expression of VEGF in each tumor tissues. Results:The healthy human peripheral blood source of DC,analog tumor cells secrete cytokines(add bombesin) inhibited DC,add Theanine cultivate,the CD40,CD80,CD83,CD86 and HLA-DR expression of DC surface,detected by flow cytometry after 7 days,Theanine can increase the CD40,CD80,CD83,CD86 and HLA-DR expression with different concentrations of theanine(25μmol/ L,50μmol/L,100μmol/L,200μmol/L,400μmol/L) deal with DC,the results of a dose-dependent manner.Theanine,and the best concentration is 200μmol/L,when the concentration is greater than 200μmol/L(such as 400μmol/L) then the CD40,CD80,CD83,CD86 and HLA-DR expression reduced.DC cultured in the first 7 days to observe the post-theanine inhibited bombesin on DC apoptosis with bombesin group,in treatment group,Theanine can inhibite to DC apoptosis.The apoptosis rate are:bombesin group is 16.0%;Theanine DC treatment group is 8.4%;PBS group is 4.0%. Theanine stimulate bombesin-DC that is able to promote secretion of IL-12,ELISA results showed that without making any deal with DC(ie bombesin group) IL-12 secretion is very little volume(63.5±3.6pg/ml),Theanine and normal group DC can secrete high levels of IL-12,200μmol/L,when the concentration of 90.4±4.9pg/ml,the test results with the anti-apoptosis results together prove that theanine can be bombesin antagonist on the DC that inhibit the function of the promotion of DC maturation,and there is a concentration-dependent manner.Theanine treatment of DC scanning electron microscope photographs showing the morphological characteristics of more mature: DC cultivate to 7 days to collect,Theanine,and the normal group of DC surface can be seen a very long process,which is characteristic of mature DC;and done deal with the DC(ie bombesin group) did not even have a very short neurite processes.Theanine on dendritic cells COX-2 expression of the mRNA.and protein Theanine treatment of different concentrations of DC 7 days,RT-PCR detection of COX-2 expression,as the drug concentration increased,the expression decreased dose-dependent manner.Dendritic cells to expression of COX-2 protein, then different concentrations of Theanine bombesin-DC 7 days deal with Western-blot detection of COX-2 protein expression,results showed that expression of COX-2 protein was inhibited and with the increase in drug concentration,inhibitory effect significantly.HLA-DR,CD86,CD83, CD80,CD40 expression of the DC were detected by flow cytometry after 7 days.PBS as control,Theanine can increase HLA-DR,CD86,CD83, CD80,CD40 expression,namely,Theanine can promote the phenotype maturation of DC and Theanine dose-dependent manner,and the best stimulation concentration of Theanine is 200μmol/L,DC regulator T cells through the suppression SCID mice experiments,intraperitoneal injection of peripheral blood lymphocytes of all immune reconstitution,the SCID mice tumorigenicity experiment,in A549 group SCID mice surface metastases in lung tissue of nodules were found lots of nodules,some of the nodular was mutually integrated,nodular central necrosis can be seen.But the DC group can't be seen.In the light microscope line histopathological examination,all visible tumor tissue was entity-like structure or glandular cavity-like structure,tumor cells long spindle,nuclear,and nucleolar clearly shows mitotic cytoplasm micro alkalophilic slightly transparent,cell size,cells were separated by fibrous tissue into cancer nests of varying sizes,which can be seen glandular whose cavity like structure.Induced anti-tumor effect of immune protection experiments,Theanine DC group to stimulate the growth of the incubation period of the extension of tumor,survival time is longer than the control group,tumor size,weight is smaller than the control group.The role of anti-tumor therapy experiments,observation of the survival time of tumor growth is longer than the control group,tumor size,weight is too,Histopathological immunohistochemical detection, observation of tumor tissues in each group the expression of VEGF compared with the control group.The results showed that theaine stimulated DC significantly inhibited the transplanted tumor in SCID mice and inhibites expression of VEGF,Histopathological immunohistochemical detection,observation of tumor tissues in each group the expression of VEGF compared to the situation.The results showed that DC stimulated theanine significantly inhibited the SCID mice transplanted tumor cells expressed VEGF,immunohistochemical detection of VEGF expression in the tumor,analysis showed that brown granules mainly localized in the cytoplasm,VEGF in PBS,Theanine Group Cancer tissues with high expression,while stimulation at theanine and/or DC group reduced the expression of tumor tissue,the total points of expression of VEGF:to stimulate the DC group＜DC group＜Theanine group＜PBS group,Comparing theanine,PBS no difference between groups,there is significant difference others.Conclusion:1.In this experiment the successful separation,amplification and differentiation of dendritic cells from the health of human peripheral blood mononuclear cells,is induced to a large number of mature dendritic cells in vitro generation,which is an important way.GM-CSF is the most fundamental cytokine of the generation and maintenance of dendritic cells development,IL-4 inhibit neutrophil and macrophage cells, promoting monocyte to dendritic cell transformation and maintenance of dendritic mature dendritic cells2.Set up a bombesin suppressed DC model which analogs lung cancer cells cytokine secretion inhibitory effect on DC under the lung cancer environment.Wich are used to improve the model is successful.3.Theanine can stimulate the bombesin to inhibit the maturation of DC and inhibit the effect that is bombesin to DC apoptosis.3.Observed theanine on the suppression by bombesin in the form of dendritic cells,apoptosis,function,COX-2 expression,theanine may be by inhibiting the expression of COX-2 to reduce endogenous Synthesis of IL-10,to promote the mature dendritic cells,thereby promote the secretion of IL-12,regulate the balance of IL-10/IL-12 through this mechanisms.4.SCID mice is the sever cellular and humoral immune deficiency, the intraperitoneal injection of T cells,the successful implementation of immune reconstitution.Intraperitoneal injection of A549 cells, successfully established model of lung adenocarcinoma.5.Using A549 line inoculated into SCID mice and stimulating DC cells by theanine.Observation of the SCID mice transplanted tumor,the immune protection and the immune therapeutic effects of metastatic tumor model is to prove the protection and activation effects of theanine on inhibited DC which is under tumor environment,and further confirms the mechanism that theanine regulats T lymphocytes through dendritic cells to inhibit lung adenocarcinoma(A549).theanine stimulate DC can inhibit the expression of VEGF,thereby inhibite the growth of tumor angiogenesis and the metastasis of tumor...