| Objective To investigate the regulatory effect of long-chain non coding RNA 028466(lncRNA 028466)on Th1 and Th2 cytokines expression in CD4~+T lymphocytes during the immune process induced by Echinococcus granulosus antigen P29(rp29)in mice.Methods1.Purification and identification of rP29:rP29 was purified by affinity chromatography and identified by Western blot.2.To establish rP29 immune model and prepare the lymphocytes of each group:6-8 w female BALB/c mice 24 in all,were randomly divided into 2 groups(control group,immune group),each group 12,the control group did not deal with;the immune group was injected with10 ug protein and the same volume Freund complete adjuvant emulsifier,after 2 w of initial immunization,the same dose was used to enhance immunity,strengthen the immunization with Freund incomplete adjuvant,and the two immunization was 3 subcutaneous injection of the abdominal subcutaneous,the total dose was 100 ul,then the second times after immunization was 2 w,the spleen cell suspension was prepared from sterile environment and lymphocytes in each group were isolated.3.To verify the relative expression of lncRNA028466 in spleen lymphocytes:(1)Real time fluorescence quantitative PCR(qRT-PCR)was used to detect the expression of lncRNA028466 in lymphocytes.(2)The lymphocyte subsets CD4~+T,CD8~+T and B lymphocytes were selected by flow cytometry.(3)The expression of lncRNA028466 was detected by qRT-PCR.4.Construction of lncRNA028466 lentivirus overexpression vector to intervene Naive CD4~+T cells:(1)lncRNA028466 lentivirus overexpression vector was obtained by packaging the lentivirus with 293T cells and transfected into naive CD4~+T cells,which were divided into overexpression group(pCDH-028466 group)and control group(vector group).The expression of IL-2,IFN-γ,IL-4 and IL-10 mRNA was detected by qRT-PCR.(2)The intracellular expression of cytokines IL-2,IFN-γ,IL-4 and IL-10 were detected by flow cytometry.(3)The secretion of cytokines IL-2,IFN-γ,IL-4 and IL-10 were detected by ELISA.5.Synthetic siRNA028466 interferes with Na?ve CD4~+T cells:(1)siRNA028466 interference fragments:siRNA1,siRNA2 and siRNA3 were synthesized by the reagent company and transfected into naive CD4~+T cells,which were divided into interference group 1(siRNA1 group),interference group 2(siRNA 2 group),interference group3(siRNA 3 group)and negative control group(negative control group).The interference effects of the three fragments were verified by qRT-PCR.(2)Naive CD4~+T cells were transfected with siRNA1 with better interference effect.The expression of cytokines IL-2,IFN-,IL-4 and IL-10 mRNA was detected by qRT-PCR.(3)The intracellular expression of cytokines IL-2,IFN-γ,IL-4 and IL-10 were detected by flow cytometry.(4)The secretion of cytokines IL-2,IFN-γ,IL-4 and IL-10 were detected by ELISA.Results1.The molecular mass(Mr)of rP29 protein induced and expressed by the genetically engineered strain purified by affinity chromatography was 31 000.2.To verify the relative expression of lncRNA028466 in spleen lymphocytes:(1)qRT-PCR results showed that the relative expression of lncRNA028466 in the lymphocytes of the immunized group was significantly lower than that in the control group(P<0.0001).(2)The proportion of CD4~+T,CD8~+T and B cells in splenic lymphocyte subsets determined by flow cytometry were 21.6%,7.1%and 56.7%in the control group,26%,8%and 56%in the immunization group.(3)The results of qRT-PCR showed that the relative expression of lncRNA028466 in CD4~+T ymphocytes of the immune group was significantly lower than that of the control group(P<0.01),and the expression of lncRNA028466 in CD8~+T and B lymphocytes was not statistically significant(P>0.05).3.Construction of lncRNA028466 lentivirus overexpression vector to interfere with Naive CD4~+T cells:(1)The results of qRT-PCR showed that the relative expression of IFN-γand IL-2 mRNA in pCDH-028466 group was lower than that in vector group(P<0.001,P<0.001),while the relative expression of IL-4 and IL-10 mRNA in pCDH-028466 group was higher than that in vector group(P<0.01).(2)The results of flow cytometry showed that the expression of IFN-γand IL-2 in pCDH-028466 group was lower than that in vector group(P<0.05),while the expression of IL-10 and IL-4 in pCDH-028466 group was higher than that in vector group(P<0.05).(3)The results of ELISA showed that the levels of IFN-γand IL-2 in pCDH-028466group were lower than those in vector group(P<0.01,P<0.0001),and the levels of IL-4 and IL-10 in pCDH-028466 group were higher than those in vector group(P<0.001,P<0.05).4.Synthetic siRNA028466 interferes with Naive CD4~+T cells:(1)The results of qRT-PCR showed that the relative expression of lncRNA028466 in siRNA 1 group was significantly lower than that in negative group(P<0.0001),but there was no significant difference between siRNA 2 and siRNA 3 group and negative group(P>0.05),indicating that siRNA 1 interference effect was better,siRNA 2 and siRNA3 had no interference effect,so siRNA 1 was used in subsequent experiments.(2)The results of qRT-PCR showed that the relative expression of IFN-γand IL-4 mRNA in siRNA 1 group was not statistically significant compared with that in negative group(P>0.05),the relative expression of IL-2 mRNA was higher than that in negative group(P<0.001),while the relative expression of IL-10 mRNA was lower than that in negative group(P<0.001).(3)The results of flow cytometry showed that the expression of IFN-γand IL-2 in siRNA1 group was higher than that in negative group(P<0.05,P<0.01),while the expression of IL-4 and IL-10 in siRNA 1 group was lower than that in negative group(P<0.05).(4)The results of ELISA showed that there was no significant difference in IFN-γand IL-4 levels between siRNA 1 group and negative group(P>0.05).The level of IL-2 in siRNA 1group was higher than that in negative group(P<0.05),and the level of IL-10 was lower than that in negative group(P<0.0001).ConclusionlncRNA028466 was down regulated and over expressed in CD4~+T cells of rP29 immune group lncRNA028466 can inhibit the expression of Th1 related cytokines IL-2 and IFN-γ,while silencing lncRNA028466 can promote the expression of Th1 cytokines IL-2 and Th2related cytokines IL-10.Therefore,lncrna028466 can participate in P29 induced host immunity by regulating the expression of Th1 and Th2 cytokines in initial CD4~+T cells Protection process. |
PDF Full Text Request