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Objective To investigate the inhibitory effect and underlying mechanism of mesenchymal stem cell (MSC) derived from different sources on follicular helper T cell (Tfh cell). Methods Umbilical cord-derived MSC (UC MSC), bone marrow-derived MSC (BM MSC) and fat-derived MSC (Fat MSC) were co-cultured with peripheral blood mononuclear cell (PBMC) for 48 h. A control group was established. Flow cytometry was adopted to calculate the proportion of Tfh cells among the lymphocytes in four groups. The content of interleukin (IL)-21 in the supernatant was detected by enzyme-linked immune absorbent assay (ELISA) in four groups. BM MSC was co-cultured with PBMC, and supplemented with indoleamine 2,3-dioxygenase (IDO) inhibitor 1-methyl tryptophan (1-MT), IL-10 antibody, human leukocyte antigen (HLA)-G antibody in the 1-MT group, IL-10 inhibition group, HLA-G inhibition group and BM MSC group without addition of other substances. After 48 h culture, flow cytometry was used to detect the percentage of Tfh cells among lymphocytes. Results Flow cytometry demonstrated that compared with the control group, the proportion of Tfh cells in the BM MSC group was significantly decreased (P<0.05). Compared with the BM MSC group, the percentage of Tfh cells in the UC MSC and Fat MSC groups was significantly higher (both P<0.05). ELISA revealed that compared with the control group, the IL-21 content in the BM MSC group was significantly decreased (P<0.05). Compared with the BM MSC group, the IL-21 contents were considerably higher in the UC MSC and Fat MSC groups (both P<0.05). The analysis of underlying mechanism revealed that the proportions of Tfh cells in the 1-MT, IL-10 inhibition and the HLA-G inhibition groups were (1.75±0.07)%, (1.31±0.09)% and (1.50±0.03)%, respectively, which were significantly higher than (1.03±0.43)% in the BM MSC group (all P<0.05). Conclusions BM MSC exerts the highest inhibitory effect upon the differentiation of Tfh cell and IL-21. The mechanism underlying suppressing the differentiation of Tfh cells differentiation is probably correlated to promoting the secretion of IDO.
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Objective:To study the expression of IL-21/BCL-6/Blimp-1 in CE patients and discusse the mechanism of the pathogenesis of the echinococcosis.Methods:27 patients and 30 health persons were collected from the first affiliated hospital of Xinjiang medical university in the same period.IL-21 was detected by ELISA and the expression of IL-21/BCL-6 /Blimp-1 mRNA was detected by Real-time fluorescence quantitative PCR (qRT-PCR) in CE patients.At the same time,17 patients were followed up in the group of patients,and the expression of IL-21/BCL-6/Blimp-1 was detected before and after treatment.Results:(1) The results of PCR showed that the levels of IL-21/BCL-6 mRNA were significantly increased in the peripheral blood mononuclear cells of the CE patients compared with the healthy control group (P<0.05).The expression of IL-21/BCL-6 /Blimp-1 mRNA in patients before the treatment was higher than that of patients after treatment(P<0.05).(2)The level of IL-21 in peripheral blood of CE patients was sig-nificantly higher than that in the healthy control group and basically returned to normal after the cure (P<0.05).IL-21 was positively correlated with BCL-6(r=0.733, P<0.01).Conclusion:BCL-6 and Blimp-1 May promote the human immune system to resist parasitic infection in the course of the development of the disease.IL-21, BCL-6 and Blimp-1 are significantly reduced after effective treatment,indicating that these factors are involved in the immune mechanism of the development of the disease.
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Objective:To express recombinant protein mIL-21-hIgGFc in 293E cells,and investigate its effect on CD8+T cell.Methods:Total RNA was extracted from the mouse spleen cells ,and then IL-21 gene was amplified by RT-PCR and inserted into expression vector PTT3-hIgGFc.PTT3-mIL-21-hIgGFc were transfected into 293E cells by calcium phosphate method.The supernatants were collected at 48 hours and 72 hours and concentrated by MOLLIPORE Labscale TM TFF system ( 5 kD membrane ).The mIL-21-hIgGFc fusion protein was purified with HiTrap TM Protein G column.The protein was quantified by SDS-PAGE and ELISA.The biological activity of the protein was determined by detecting the change of the phenotypes of CD 8+ T cells treated with the protein.Results: The constructed recombinant plasmid PTT 3-mIL-21-Fc was confirmed by sequencing.PTT3-mIL-21-Fc was transfected into 293E cells,mIL-21-Fc protein in culture supernatant was collected after 48 hours and 72 hours.The protein in cell su-pernatant reached a concentration of 787 ng/ml which was determined by ELISA.The protein was purified by Protein G chromatography column.P1A-specific T cells were treated with mIL-21-hIgGFc, and found that the CD44low CD62Lhi CD8+ population increased compared to the control.Conclusion:We built PTT3-mIL-21-hFc recombinant plasmid, expressed mIL21-hFc fusion protein in 293E cells,and purified by Protein G column.By treating mIL-21-hFc ,the antigen-primed CD8+T cells prefer to differentiate into CD44low CD62Lhi CD8+T cells which had been reported as a memory stem phenotype .This protein may be used to improve the effectness of adoptive T cell cancer therapy.
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Objective:To construct Interleukin 21 (IL 21) expression plasimd pCDNA3.1 IL21 and express it in Hela cell and analysis its acitivity of costimilating T cell proliferation with anti CD3 monoclonal antibody in vitro.Methods:The CDs of IL 21 was amplified by PCR using the pMD IL21 as templet.The expression plasimd pCDNA3.1 IL21 was constructed by inserting the sequence of coding region of the IL 21 into pCDNA3.1/Hismyc B containing CMV promoter and transfected into Hela cells.The stability expression stain was screened by the condition media containing 400 mg/L G418 and cloned through the limited dilution method.The target protein was purified through Ni 2+ chelating Sepharose Fast Flow.The bioactivity was confirmed by MTT method on costimulating the T cell proliferation with anti CD3.SDS PAGE and Western blot analysis the rhIL 21 expressed.Results:hIL 21 was expressed in Hela cell successfully.SDS PAGE analysis showed the IL 21 fusion protein with Mr 18 000 or so was expressed in supernatant of the cells.The rhIL 21 has significant proliferation effect on mature human T cells in the presence of anti CD3 monoantibody.Conclusion:The rhIL 21 with bioactivity has been obtained,which may help researcher study its new function and effects. [