Study On The Effect Of HLA-G On Trophoblast Cell Multiplication

Background:The HLA-G gene is a kind of non-classical human leukocyte antigen(HLA)class 1b genes,located on the sixth short arm of chromosome of the human being,In contrast to classical HLA classⅠantigens,the HLA-G exhibits selective tissue distribution;limited molecular polymorphism;and alternative transcription of spliced mRNAs that encode at least seven different HLA-G isoforms.Early studies suggested that the HLA-G only express on extravillous trophoblast cells of the maternal-fetal interface in normal pregnancy Now it has been found the HLA-G also expresses on the vascular endothelial cells of the fetal chorionic villi,the amniotic fluid,the trophoblast proliferative cells at the basal layer of the villus and the epithelial of the thymic etc.The pregnancy is considered successful allograft.It is an important role for the HLAG to tolerate the maternal-fetal immune and maintant the normal pregnancy.It is due to the expression on the maternal-fetal interface;A lot of pregnancy-associated diseases relevant with the trophoblastic dysplasia,abnormal infiltration and the undesirable formation of the placenta.Numerous studies shew that some pregnancy-associated diseases relevant with the abnormal expression of the HLA-G.It plays an important role at maternal-fetal immune tolerance,embryo division and implantation,etc.The mechanism is by inhibiting uterine decidual natural killer cells(dNK)and cytotoxic T cell(CTL), regulating the balance of cytokine release,improveing embryo cleavage rate,etc. Thus the HLA-G may have the function of regulating trophoblast proliferation and differentiation,while protecting these cells from maternal immune cells attack.The study of the HLA-G is a relatively new field,our research has important theoretical and clinical value.This study may have found the new features about the HLA-G.It has an essential significance to understand deeply with the mechanism of the immune tolerance of the pregnancy and the pregnancy-related diseases and the pathogenesis of the pathological pregnancy .And our study may have potential clinical value for in vitro fertilization.RNAi(RNA interference)is the process that the Exogenous and endogenous double-stranded RNA in cells induce to inhibit the expression of the homologous genes.This phenomenon was observed in a series of organisms and the cultured mammalian.Small interfering RNAs(small interfering RNAs, siRNAs)are RNAi-mediated effector responses.RNAi technology can remove or turn off specific expression of particular genes,so it has been widely used to explore gene function and gene therapy to infectious diseases and malignant tumors.Objective:Construct adenovirus vector which can transcribe siRNA specially targeting HLA-G mRNA.Transient transfect HLA-G high expression cell line JEG-3 to knock-down HLA-G expression.To construct HLA-G low expression trophoblasts model in vitro.To detect the infection efficiency of the adenovirus vector transfecting JEG-3 cells by X-Gal staining.And with HLA-G low expression cell we further study how HLA-G regulat cell proliferation.Methods:Using RNAi technology,the HLA-G high expression cell line JEG-3(derived from human chorionic cells)was used for trophoblasts function studies.Through adenovirus vector transiently transfected into JEG-3 cells to set up HLA-G low expression in trophoblast cell model.1)through X-Gal stain we detect the infection efficiency of adenovirus vector;2)expression of HLA-G in Ad vector transfected JEG-3 cells was checked by RT-PCR and Western blot. Then the JEG-3 cell was studied by non-radioactive cytotoxicity assay.Detected OD value of 490nm,The OD value can represent the relative quantity of cell proliferation.Results:①The Ad vector we have constructed can transfect JEG-3 cells with the efficiency of 100%at the MOI of 100 and induce significant RNAi in these cells.HLA-G expression in JEG-3 cells was successfully knocked-down on both mRNA and protein level;②Then the cells with HLA-G gene hyp-express was studied by application of a nonradioactive cytotoxicity assay.Results:The non-specific siRNA group than the control group,the difference was not significant(q=3.15,P＞0.05);the purpose siRNA group than the control group, the difference was statistically significant(q=5.58,P＜0.01);the purpose siRNA group than the non-specific siRNA group,the difference was statistically significant(q=3.92,P＜0.05).Conclusions:We Constructed the adenovirus vector who could interferent specificly the HLA-G gene express time in our experiment,and transfected human trophoblast cells,set up a vitro research model.This study reveals an important nonimmune function for HLA-G in controlling trophoblast cell multiplication.