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Objective To investigate the effect of Trichomonas vaginalis macrophage migration inhibitory factor (TvMIF) on THP-1 macrophages.. Methods Recombinant TvMIF protein was prokaryotic expressed and purified, and endotoxin was removed after identification. Following exposure to TvMIF at concentrations of 0, 1, 5, 10, 50 and 100 ng/mL, the cytotoxicity of the recombinant TvMIF protein to THP-1 macrophages was tested using cell counting kit (CCK)-8 assay, and the apoptosis of THP-1 macrophages and reactive oxygen species (ROS) were detected using flow cytometry. The relative expression of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), caspase-1, interleukin-1β (IL-1β) and IL-18 genes was quantified using real-time fluorescent quantitative PCR (qPCR) assay, and the expression of caspase-1, NLRP3, gasdermin D (GSDMD), gasdermin D N-terminal (GSDMD-NT) and pro-IL-1β proteins were determined using Western blotting assay. Results Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) displayed successful expression and purification of the recombinant TvMIF protein with a molecular weight of 15.5 kDa, and the endotoxin activity assay showed the successful removal of endotoxin in the recombinant TvMIF protein (endotoxin concentration < 0.1 EU/mL), which was feasible for the subsequent studies on protein functions. Flow cytometry revealed that the recombinant TvMIF protein at a concentration of 10 ng/mL and less promoted the apoptosis of THP-1 macrophages, and the highest apoptotic rate of THP-1 macrophages was seen following exposure to the recombinant TvMIF protein at a concentration of 5 ng/mL, while the recombinant TvMIF protein at concentrations of 50 and100 ng/mL inhibited the apoptosis of THP-1 macrophages. Exposure to the recombinant TvMIF protein at a concentration 1 ng/mL resulted in increased ROS levels in THP-1 macrophages. qPCR assay quantified significantly elevated caspase-1, NLRP3, IL-18 and IL-1β expression in THP-1 macrophages 8 hours post-treatment with the recombinant TvMIF protein at a concentration 1 ng/mL, and Western blotting determined increased caspase-1, NLRP3, pro-IL-1β, GSDMD and GSDMD-NT protein expression in THP-1 macrophages following exposure to the recombinant TvMIF protein at a concentration 1 ng/mL. Pretreatment with MCC950 significantly reduced GSDMD and GSDMD-NT protein expression. Conclusions High-concentration recombinant TvMIF protein inhibits macrophage apoptosis, while low-concentration recombinant TvMIF protein activates NLRP3 inflammasome and promotes macrophage pyroptosis.
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Objective To investigate the effect of long intergenic non-coding RNA COX2 (lincRNA-COX2) on apoptosis and polarization of Listeria monocytogenes (Lm)-infected RAW264.7 cells. Methods RAW264.7 cells were cultured and divided into control group (uninfected cells), Lm infection group, negative control of small interfering RNA (si-NC) group, si-NC and Lm infection group, small interfering RNA of lincRNA-COX2 (si-lincRNA-COX2) group, si-lincRNA-COX2 and Lm infection group. RAW264.7 cells were infected with MOI=10 Lm for 6 hours, and then the inhibition efficiency of siRNA transfection was detected by fluorescence microscope and quantitative real-time PCR (qRT-PCR). The expression levels of cleaved-caspase-3(c-caspase-3), caspase-3, B-cell lymphoma-2 (Bcl2), Bcl2 associated X protein (BAX), arginase 1 (Arg1), inducible nitric oxide synthase (iNOS) were detected by Western blot analysis. Results c-caspase-3/caspase-3, BAX/Bcl2 and iNOS were significantly up-regulated, while the level of Arg1 was down-regulated in Lm-infected RAW264.7 cells compared with control group. LincRNA-COX2 knockdown inhibited the increase of protein levels for BAX/Bcl2, c-caspase-3/caspase-3 and iNOS in Lm-infected RAW264.7 cells, while the level of Arg1 in Lm-infected RAW264.7 cells was up-regulated. Conclusion Knockdown of lincRNA-COX2 can inhibit cell apoptosis and suppress the macrophage polarization into M1 type in Lm-infected RAW264.7 cells.
Subject(s)
Animals , Mice , Apoptosis/genetics , bcl-2-Associated X Protein/metabolism , Caspase 3/metabolism , Cyclooxygenase 2/metabolism , Listeria monocytogenes/pathogenicity , Macrophages/microbiology , RNA, Long Noncoding/metabolism , RNA, Small Interfering/geneticsABSTRACT
Objective To construct the prokaryotic expression vector of human procalcitonin(PCT) and obtain the highly purified recombinant PCT protein.Methods PCT primers were designed based on the sequence of PCT gene from NCBI,and PCT gene was amplified by PCR.Then,the recombinant vector PCT/pET-22b(+) was constructed and transferred into E.coli BL21 to induce the expression of recombinant PCT protein.Last,the recombinant PCT protein was purified by the nickel column affinity chromatography and identified by Western blot and the colloidal gold method,respectively.Results The results of agarose gel electrophoresis showed that the product of PCR amplification was about 350 bp.The homology comparison analysis revealed that the PCT gene fragment(348 bp) was successfully inserted into pTG19-T vector,and that no any base mutation was found.When the recombinant plasmid of PCT/pET-22b(+) was digested with BamH Ⅰ and HindⅢ,two pieces of about 350 bp and 5 500 bp were obtained.SDS-PAGE showed that the recombinant PCT protein with about 14 000 of Mr was existed in soluble form,and was easily obtained by the nickel column affinity chromatography.Moreover,western blot and the colloidal gold method demonstrated that the recombinant PCT protein was successfully expressed and contained histidine label(His-tag).Conclusion The recombinant vector PCT/pET-22b(+) is successfully constructed by the recombinant DNA technology,and the recombinant PCT fusion protein is successfully obtained by the nickel column affinity chromatography.
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Objective To clone and express the thioredoxin(Trx)from RH strain tachyzoites of Toxoplasma gondii,estab?lish the prokaryotic expression vector and purify the recombinant protein,then produce the polyclonal anti?Trx antibody in rab?bits. Methods Trx fragment was amplified by PCR and cloned into the pET?28a(+)vector,and the recombinant protein was in?duced with IPTG and purified by Ni?NTA affinity chromatography. The polyclonal antibody specificity was detected by Western blotting. Results The trx gene was amplified from T. gondii cDNA by PCR. The recombinant plasmid trx/pET?28a(+)was use?fully constructed,and the recombinant TRX protein was expressed and purified. The TRX polyclonal antibody was also ob?tained. The specific band of TRX was detected by Western blotting. Conclusion Western blotting can detect the specificity of polyclonal anti?Trx antibody,which will facilitate the biological functions of Trx.
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The article is attempted to introduce PBL teaching in clinical biochemistry. The PBL teaching has improved some aspects of students' abilities in the self-study, accessing to information, solving the problem, teamwork and developing thinking. But in the course of PBL teaching, there are three problems: shortage of qualified teachers, teaching system and students' inability. This article puts forward the countermeasures to resolve these problems.
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Objectives To establish a reliable method for isolating and purifying soluble collagen type Ⅱ(SCⅡ)by optimizing preparative procedure.Method The chicken sternal cartilage was selected as raw material.Guanidine hydrochloride was used to remove the proteoglycans.The digestion manners of pepsin,sodium chloride concentrations for salting,types of DEAE anion resin were studied for extracting SCⅡ.The SCⅡ identification was made by SDS-PAGE,absorption spectrum and amino acid analysis.Result It was convenient for pre-treatments of chicken sternal cartilage.The proteoglycans could be efficiently removed by 4 mol/L guanidine hydrochloride.The satisfied results were obtained by limited enzyme digestion of pepsin added by two steps.The optimizing concentration of sodium chloride for salting was 2.4mol/L.SDS-PAGE maps revealed that the bands of purified SCⅡand standard CⅡ were at the same location.The absorption peak of SCⅡ was at 230nm.The concentrations of Gly,Pro and Ala were the highest in 15 amino acids.Conclusion The improved method has significant advantages of simple working process,result reliability and convenient source of raw material.It is suitable for purifying the SCⅡ at variable scales in research works and clinic application.The SCⅡ product obtained has high purity and accords with the characteristics of collagen type Ⅱ.
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