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Objective_To explore the influence and mechanism of IL-31 on the expression of VEGF, EGF and EG-FR in 16HBE cells.Methods_16HBE cells were cultured and treated with IL-31 with or without SB203580 or SP600125, real-time PCR and Western blot were applied to determine the mRNA and protein expression of VEGF, EGF and EGFR respectively.Meanwhile, Western blot was used to examine the changes of P38 MAPK and JNK signaling pathways.Results_Compared with control group, the mRNA expression of VEGF, EGF and EGFR was increased markedly under the stimulation of IL-31 ( P<0.01 ) , the expression of p-P38 MAPK and p-JNK signifi-cantly increased ( P<0.01) .Compared with IL-31 group, the expression of p-P38 MAPK significantly decreased in IL-31 combined with SB203580 or SB203580 group ( P <0.01 ) , while the expression of p-JNK markedly decreased in IL-31 combined with SP600125 or SP600125 group( P<0.01) .Compared with IL-31 group, the expression of VEGF was significantly decreased in IL-31 combined with SB203580 or SP600125 group ( P <0.01 ) , while the expression of EGF and EGFR was markedly declined in IL-31 combined with SB203580 group ( P<0.01 ) .Conclusions_IL-31 may up-regulate the expression of VEGF through activating P38 MAPK and JNK signaling pathways and up-regulate the expression of EGF and EGFR through activating P38 MAPK signaling path-way in16 HBE cells.
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Purpose To investigate the effect of interleukin-10(IL-10)on IL-15mRNA and IL-6mRNA in Hela cells induced by lipopolysaccharide(LPS)and to analyze their activiated signal transduction pathways.Methods Extracted total RNA and total proteins of cultured Hela cells,which were treated with different concentrations of LPS,or IL-10 alone or in combined use,were to analyze the levels of IL-15mRNA and IL-6mRNA using RT-PCR and to analyze the expression of signal transduction pathway proteins using Western blot.Results RT-PCR indicated that expression of IL-15mRNA and IL-6mRNA in Hela cells strikingly increased after 12 h using 1 ng-10μg LPS(P＜0.01 vs control),which had dose-dependence and achieved peak value using 100 ng/mL.During 0-24 h,expression of IL-15mRNA and IL-6mRNA strikingly increased with time changing(P＜0.01 vs control),which had time-dependence and achieved peak value at 12 h.Expression of IL-15mRNA and IL-6mRNA had no conspicuous difference in Hela cells treated with IL-10(10 ng/mL)alone(P＞0.05 vs control).Different concentrations of IL-10 1,10,100 ns/mL)markedly downregnlated expression of IL-15mRNA and IL-6mRNA in Hela cells induced by LPS.Furthermore,the effect of inhibition will be more obvious with dose increasing.Western blot indicated that LPS upregulated expression of IL-15mRNA and IL-6mRNA by phosphorylation of PI3K/AKT and ERK1/2.IL-10 blocked phosphorylation of AKT,but could not affect phosphorylation of ERK1/2.Conclusion IL-10 downregulated the expression of inflammatory cytokines IL-15 and 1L-6 induced by LPS,which may correlate with the fact that phosphorylation of AKT was blcoked by IL-10.Therefore,IL-10 may be used to prevent and treat some clincal infective diseases.
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Purpose To explore the antiviral effect of interleukin 29(IL-29) on hepatitis B virus in vitro.Methods To study the antiviral effect of IL-29 against hepatitis B virus by the amount of HBV mRNA detected .Through the quantity of mRNA translated from genes of MxA,2′,5′-OAS,PKR and RNase L as well as the signal pathway induced by IL-29,we used RT-PCR and Western blot to discuss the anti-hepatitis B virus mechanism which was stimulated by IL-29.Results The amount of HBV mRNA in HepG2.2.15 cells was reduced by stimulation of IL-29.The expression of MxA and 2′,5′-OAS was up-regulated,as well as P-ERK and P-AKT were activated by IL-29.Conclusion These findings showed that IL-29 had obvious antiviral activity towards HBV in HepG2.2.15 cells.
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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. [
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Objective: To clone cDNA of human interleukin-29(IL-29) and express it in cos-7 cells, and to study its anti-HBV activity in vitro. Methods: Total RNA was extracted from PBMCs which had been infected with vesicular stomatitis virus(VSV) in vitro.IL-29 cDNA was amplified using one-step RT-PCR technique. The recombinant expressing plasimd of psectagB/His-myc-IL29 was constructed by inserting IL-29 cDNA into the vector and was then transfected into cos-7 cells. Stable expression stains were screened by Hygromycin B and limited dilution method. The target protein was purified through Ni2+-chelating Sepharose Fast Flow. Anti-viral bioactivity of the recombinant IL-29 fusion protein was analyzed through an in vitro model of production of HBV by the HepG2.2.2.15 cell line.ELISA was used for detection of the viral titers in the cell cultural supernants. Results: IL-29 was cloned and stably expressed in cos-7 cells successfully. SDS-PAGE and Western blot analysis showed multiple bands of the target protein with the molecular weights between 20 000 and 33 000, and the major band was located at about 33 000, indicating the fused IL-29 modified by additional glycosylation. The rhIL-29 was shown to dose-dependently inhibit secretion of HBsAg and HBeAg accompanied by the reduction of HBV genomic DNA in the cells tested. The inhibition ratio of HBsAg and HBeAg production was attained 85% at a concentration of 160 ?g/L of rhIL-29. Conclusion: The rhIL-29 with anti-HBV activity has been obtained.