The Killing Effection And Mechanism Of NK Cell Line Transinfected NK G2D Receptor To Laryngeal Carcinoma

Objective Laryngeal cancer is the common malignant tumor in otolaryngology.Laryngeal squamous cell carcinomas comprise the vast majority(96%)of laryngeal malignancies.At present,the incidence of laryngeal cancer is increasing obviously.It is not only because of the improvement of clinical diagnostic technique,average life augment,but also have the possibility of aggravation of air pollution,smoking,and some long term occupational contacting with carcinogens.This is consistent with the phenomena of the higher incidence of laryngeal cancer in cities than in countryside,in heavy industry cities than in light industry cities.Laryngeal cancer constitute 5.7-7.6 Percent of all malignancies, 7.9-35 percent of otorhinolaryngologic malignancies.Till now,the molecular pathogenesis of carcinomas has been not clear.Of the most frequent malignancies in the United States,cancers of the larynx and of the uterine corpus are the only ones not to show an increase in 5-year survival rates over the last 30 years.The increasing use of chemo-and radiotherapy and conservative surgery to preserve organs and their functions has probably led to a better quality of life in patients with laryngeal cancer,but has definitely failed to improve survival,which remains the primary aim.So find an effective method to improve the treatment effect is the most important thing of now,using immunotherapy of NK cell may be a better candidate.NK cells constitute an important component of the innate immune system,providing surveillance against certain viruses,intracellular bacteria and transformed cells.Thus NK cells form a first line of defence against pathogens or host cells that are stressed or cancerous.NK cells exert cell-mediated cytotoxicity and act to regulate innate and acquired immune responses through the release of various cytokines (such as IFNγ,GM-CSF and TNF-β)and chemokines.Therefore,they stand as a bridge between innate and adaptive immune responses.Unlike T cells, NK cells killing of virus-infected or malignant transformed cells do not need pre-sensitization and are independent of a MHC restricted manner,thus take a vital position in both innate and acquired immunity. Therefore,NK cells are now the hot items in immunological researches and are considered as promising reagents in adaptive transferring treatment of multi-cancers.However,the existence of target cells with normal MHC class I expression thatare still sensitive to NK cells suggests that there may be other mechanisms to regulate NK cell activity.NK cells express surface receptors that receive signals from the environment and determine their response to foreign or malignant cells.The activating receptors should play a major role and are believed to be necessary for the initial activating of NK cells.The effector functions of NK cells are regulated by integrated signals across the array of stimulatory and inhibitory receptors(such as NKG2A and NKG2B)engaged upon interaction with target cell surface ligands.The well-characterized example to date is the NKG2D receptor and its ligands.NKG2D is a peculiar activating receptor that is expressed as a disulphide-linked homodimer by all NK cells, alphabeta CD8(+)T cells,gammadelta T cells and murine macrophages.It not only activates NK cells but also delivers co-stimulatory signals to CD8(+)T cells and gammadeltaT cells.In human,the NKG2D receptor binds to the stress-inducible polymorphic nonclassical MHC molecules MICA (MHC class I chain-related A),MICB and the MHC class I-related UL16-binding proteinsl,2,3(ULBP1,2,3),which are up-regulated strongly in many types of tumor cells.Recent studies reveal that the expression of MlCs and ULBPs on human tumor cells is sufficient to overcome the inhibitory effects of MHC class I expression on NK cells and indicate that NKG2D provides first line surveillance against stressed or abnormal cells that have been induced to express one of its ligands.The establishment of NK cell lines provided favorable tools for NK cell basic researches.Currently,six malignant NK cell lines(YT, NK-92,NKL,HANK-1,KHYG-1,NK-YS)have been established and sufficiently well characterized.Their immunophenotype is remarkably similar and described as followed:CD1~-CD2~+ CD3~-CD4~-CD5~-CD7~+CD8~-CD16~-CD56~+CD57~-.All these NK cell lines showed natural cytotoxicity,among which YT cell line attracted more scientists and generated much more basic data for NK cell research.YT cell line was derived from an acute patient with lymphoma and thymoma in 1985.YT has typical surface marker of NK cells and is independent or low dependent on IL-2 for cytotoxicity and proliferation.We can only detect the mRNA expression of NKG2D, but not detect the protein expression in YT cells because of long period culturing in labortary.YT has low cytotoxicity compared with NK-92 and NKL.We try to clarify the role of NKG2D on NK cell development and cytolytic mechanisms by transfecting NKG2D into YT cells.The expressions of the major cellular ligands for human NKG2D in Laryngeal neoplasms are still unclear.If the MICA and ULBPs were highly expressed,it will be very useful To study the adoptive immunotherapy. So first we analyzed the gene expression of MHC class I-related chain A (MICA)and UL16 binding proteinl,2,3(ULBPs)in tissues of laryngeal cancer and HEP-2 cell line by means of revearse transcription polymerase chain reaction(RT-PCR).Then we plan to transfect NKG2D into YT cells by gene engineering technology to observe the killing effect on Hep2 cell line.Methods(1)Detected the gene expression of MICA and ULBP-1,2,3 as the major cellular ligands for NKG2D in Laryngeal cancer and Hep2 cell line by means of revearse transcription polymerase chain reaction(RT-PCR).(2) Construction of NKG2D clone vector.A NKG2D gene fragment,with a length of about 650bp,was amplified from the NK-92 cell line by RT-PCR and was sequenced by cloned toplasmidpGEM-TEasyvector.(3)Construction of NKG2D eukaryotic expression vector.The recombinant plasmid pGEM-T Easy/NKG2D was digested with EcoRI and BamHI,then NKG2D fragment was isolated and inserted into the corresponding restriction site on eukaryotic expression vector pEGFP-N1.(4)The lipofectamine was used to transfect recombinant eukaryotic expression plasmid(pEGFP-N1-/NKG2D) into the CHO cells.The expression level of.NKG2D gene in transfected CHO cells was detected by RT-PCR,and the expression level of NKG2D protein was detected by fluorescence microscopy,western blot and immunohistochemical staining.(5)Recombinant eukaryotic expression vectors were transfected into YT cells and detected how its biological function changed.Recombinant vectors were transfected into YT cells with Lipofectine.Proliferation assay and cytotoxicity analysis were performed as MTT method.Cytotoxic molecules levels such as perforin and TNF/TNFR superfamily were assayed by RT-PCR withβ-actin as a control. Results1.expression of MICA and ULBP-1,2,3In this study,we analyzed the gene expression of MHC class I-related chain A(MICA)and UL16 binding protein-1,2,3(ULBPs),the major cellular ligands for human NKG2D,in laryngeal cancer and Hep2 cell line.We show that MICA and ULBP-3 are often strongly coexpressed by laryngeal cancer and Hep2.while the ULBP1,2 are relatively lower in Laryngeal cancer by means of revearse transcription polymerase chain reaction(RT-PCR).2.Cloning of full length NKG2D cDNA.Total RNA was isolated from fresh culturing NK-92 cells.Reverse transcription was performed on 5ug of total RNA with an ologo(dT)primer. PCR was used to amplify specific primers for NKG2D.After PCR product was detected by ethidium bromide-stained agarose gel electrophoresis, NKG2D was then ligased with pGEM-T Easy vector,cloned and sequenced, which confirmed that the PCR product was cDNA of NKG2D.The sequence of cloned NKG2D cDNA was identical with with those in Gene Bank.3.Construction of the vector pEGFP-N1/NKG2DThe NKG2D fragment was digested by EcoR I and BamH I from pGEM-T Easy/NKG2D,and was cloned into corresponding sites of the pEGFP-N1 also.Then,the recombinant vector was transformed to competent cell HB101.The postive clones which were screened by PCR and identified by restriction endonucleases were the recombinant expression vector of pEGFP-N1/NKG2D.4.Expression of NKG2D in CHO cellsThe recombinant vector pEGFP-N1/NKG2D was transfected into CHO cell lines by lipofectamine method.After cultured in selective medium, G418 resistant clones were selectedand transcription levels of NKG2D were identified by RT-PCR.The green fluorescence could be seen in transfected CHO cells under fluorescence microscope.Western blot and Immunohistochemical staining detection showed that NKG2D expressed in transfected cells.5.Transfection of recombinant eukaryotic expression vector pEGFP-N1/NKG2D into YT cells and its influence on biological functions of YT cells.After transfected with NKG2D,the proliferation effect of YT cells was invariable,but the cytotoxicity to Hep2 cells increased significantly.Transcription levels of IFNγ,IL-2,TNFα,perforin and TWEAK show remarkable increase.Conclusions(1)the gene expression of MICA and ULBP-3 are frequently coexpressed strongly by laryngeal cancer and HEP-2.while the ULBP1,2 are weakly expressed.(2)We acquired the full length of NKG2D gene fragment and constructed recombinant eukaryotic expression vector pEGFP-N1/NKG2D by gene engineering technology.(3)Transcription levels of NKG2D were identified by RT-PCR in transfected CHO cells. The green fluorescence could be seen under fluorescence microscope. Western blot and Immunohistochemical staining detection showed that NKG2D truly expressed in transfected cells.(4)The transfection of NKG2D into YT cells enhanced their cytotoxicity on Hep2 cells. Transcription levels of IFNγ,IL-2,TNFα,perforin and TWEAK show marked increase.(5)NKG2D gene can be used to modify YT cell line for adaptive cellular immunotherapy.Collectively,although cancers can not be cured till now,laryngeal cancer is still one of the tumors that easily cause human immune response, which is probably another explanation that laryngeal cancer patients often have a relative good survival rate besides the Thl shift expression. YT cells can be used as a tool for Studying of NK cell development and cytotoxic mechanisms and explaining regulatory mechanism of cytokines involved in NK cell biologic activity.In addition,NK cell lines can be modified with NK receptor genes to increase the availability for clinical adoptive cellular immunotherapy.