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OBJECTIVES@#To establish an experimental method for evaluating material permeability of type I collagen hydrogels.@*METHODS@#Using BSA-FITC as an indicator, by combining BSA-FITC with PBS they were used as permeability media, and using transwell load hydrogen sample to detect BSA-FITC transparent rate.@*RESULTS@#In the concentration range of 100 μg·mL~0.781 μg·mL, the standard curve ≥ 0.99, Lower Limit of Quantity (LLOQ) is 3.125 μg·mL, RSD <5%, detection recovery rate is in the range of 80%~120%.@*CONCLUSIONS@#In this study, we established an experimental method for evaluating material permeability of hydrogel. The BSA-FITC transparent rate of type I collagen hydrogel was 100% at 28 h.
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Collagen Type I , Chemistry , Hydrogels , Chemistry , Materials Testing , PermeabilityABSTRACT
Objective To investigate the influence of extracellular high mobility group box 1 (HMGB1) on viral replication in HTLV-1 infected T cells.Methods HMGB1 in culture supernatants of adult T-cell leukemia virus 1 (HTLV-1) virus-negative cell:Jurkat,MOLT4 cells and HTLV-1 virus-positive cells:MT2,MT4,was detected by ELISA;The HTLV-1 long terminal repeat reporter gene (pHTLV-1-LTR-luc) was transfected into MT2 cells by Tfx-50-mediated transfection,and 0.25,0.50,0.75 μg/ml of HMGB1 polyclonal antibody(HMGB1 PcAb) and its isotype control rabbit IgG antibodies,0.03,0.1,0.3 μg/ml rhHMGB1 and its control PBS,were added into culture supernatant respectively,then luciferase activity was detected after 48 h;Similarly,0.25 μg/ml HMGB1 PcAb and the isotype control antibody,0.3 μg/ml rhH-MGB1 and the control PBS were added to the culture supernatant of MT2 cell,the viral gene,pol1,pol2,gag,env,etc,were performed by real-time PCR.Results Culture supernatant HMGB1 levels has no significant difference between HTLV-1 positive cells MT2 and MT4 and the other two virus-negative T cell lines;Compared with isotype control antibody group,the culture supernatant,to which is added 0.25 μg/ml HMGB1 PcAb,can significantly inhibit the HTLV-1-LTR transcriptional activity and suppress the expressions of the viral gene pol1,pol2,gag,env.Compared with the control PBS,0.3 μg/ml rhHMGB1 significantly promotes the transcriptional activity of the HTLV-1-LTR and the expressions of the viral gene pol1,pol2,gag,env.Conclusion The extracellular HMGB1 can promote viral replication of HTLV-1 infected T cells.
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Objective To explore the effect of HTLV-1 (human T-cell leukemia virus type 1 ) Tax protein on the DcR3 gene expression in T cells.Methods The construction of DcR3 (-1010 bp to +114 bp) luciferase reporter gene; MT2,TaxP,and Jurkat E6-1 cells were transfected with DcR3 luciferase reporter gene (pGL3-DcR3-1uc) using liposomes according to the manufacturer's instructions.For the control group,pGL3-basic replaced it respectively.At 48 h after incubation,luciferase activity was measured with a luciferase assay system; Jurkat cells were transfected with pCMV-Tax-Bam using liposomes,and total RNA was extracted from the cells at 48 h after incubation.Reverse transcription was performed using standard protocols.Then real time PCR with primers DcR3 and 3-actin was conducted;The expression of DcR3 protein was detected by flow cytometry in MT2,TaxP,and Jurkat cells.Results The construction of DcR3 luciferase reporter gene is identified correctly; The detection of luciferase activity showed that the luciferase activity of experimental group in MT2 cells was increased by (32.07±12.43)-fold,of which in TaxP cells was increased by ( 13.27±4.04)-fold,and the luciferase activity of experimental group in Jurkat cells was increased by ( 1.26±0.49 ) -fold.And there is statistic significance about the relative luciferase activity of experimental group in MT2 cells and TaxP cells compared to the relative lueiferase activity of experimental group in Jurkat cells respectively ( P＜0.01 ) ; The result of real-time PCR showed that the level of DcR3 mRNA in the experimental group was higher compared with the control group(P＜0.05) ; The result of FCM shows the expression of DcR3 protein in MT2,TaxP cells is higher than that in Jurkat cells( P＜0.05 ).Conclusion Tax protein can promote the expression of DcR3 gene in T cells.