ABSTRACT
Objective To construct a eukaryotic expression vector pEGFP N1/IL-37b of full-length and mature IL-37b,and to detect the expression of both full-length and mature IL-37b in RAW 264.7 cells, a mouse macrophage cell line. Methods To construct the eukaryotic vectors of full-length and mature IL-37b by using plasmid pUBC/IL-37b as a template containing the coding region of IL-37b full-length gene. To detect the expression of IL-37b by western blot and confocal microscopy after transfected the recombinant plasmid into RAW 264.7 cells, and to detect the inhibition of full-length and mature IL-37b on IL-6 production by real-time PCR. Results Eukaryotic vectors pEGFP N1/IL-37b expressed full-length and mature IL-37b after transfection in cells, which inhibited LPS-induced IL-6 production. Conclusions Eukaryotic vectors of full-length and mature IL-37b can be successfully constructed,and lays a foundation for further study of anti-inflammation functions and mechanisms of IL-37b.
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Objective To establish a patient-derived xenografts (PDX) mouse model of liver cancer (LC) and to explore its role in precision medicine.Methods PDX model was established by subcutaneous implantation of tumor tissues in NCG mice.The morphological structure of tumor tissue was exaimed using HE staining.Fifteen BALB/c nude mice were subcutaneously inoculated with tumor cell suspension from the PDX models.The xenograft mice were randomly divided into 5-fluorouracil (5-FU) group, sorafenib group and negative control group.The tumor volume and body weight of the tumor-bearing mice were measured regularly, the tumor inhibition rate was calculated and the curative effect was evaluated.Results The success rate was 33.3% (6/18) in the establishment of liver cancer PDX mouse model, and the model well retained the characteristics of the primary tumor.In one case of PDX mouse model, the tumor inhibition rates of 5-FU and sorafenib group were 63.7% and 29.6%, with a statistically significant differece between them (P< 0.05), and there was no significant difference between the sorafenib group and negative control group, consistent with clinical observation.Conclusions The PDX mouse model of liver cancer can maintain the histological structure of primary tumor, and can be applied to precision medicine for patients with liver cancer.
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Objective To establish lentiviral expression vectors for Smurf1 silencing and assess the effects of Smurf1 silencing on cell migration.Methods HeLa and A549 cells were infected with lentiviral expression vectors for Smurf1 silencing respectively.After 7 days,the stable cell lines with Smurf1 silencing were obtained after puromycin-resistance screening,enrichment and expansion.The intracellular gene and protein levels of Smurf1 were detected by qPCR and western blot.Transwell assay was used to assess the effect of Smurf1 silencing on cell migration.Results The stable cell lines with Smurf1 silencing are constructed successfully.Silencing of Smurf1 down-regulated cell migration rate detected by Transwell assay.Conclusion Smurf1 promotes cell migration.
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Objective To investigate the expression of recombinant IL-37b protein and removal of the endotoxin, and identify its biological activity.Methods The prokaryotic expression vector pET28/IL-37b was constructed and to transform Escherichia coli (E.coli) Rosetta.After induction with IPTG, the recombinant protein was purified through Ni2+-NTA gel column and identified by SDS-PAGE and Coomassie brilliant blue staining.Then, the endotoxin protein was removed and was treated with LPS-stimulated RAW 264.7 cells.The culture supernatant was collected.The expression of IL-6 was detected by ELISA and the biological activity of the protein was identified.Results The recombinant IL-37b with high purity was expressed and the endotoxin produced by prokaryotic expression was reduced, and it was identified to have good biological activity.Conclusions In this study a recombinant IL-37b protein with high biological activity is successfully obtained.
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Objective To investigate the effect of Smad3 on cell migration of A549 and HeLa cells.Methods Primers for pCMV-Myc-Smad3 plasmid construction and siRNA targeting Smad 3 were designed and synthesized .pCMV-Myc-Smad3 plasmid was constructed with molecular cloning techniques .Overexpression of Smad 3 with Myc-tag or silencing of endogenous Smad3, and then scratch assay was used to detect the migration ability of A 549 and HeLa cells in vitro. Results pCMV-Myc-Smad3 plasmid was successfully constructed .Overexpression of Smad 3 significantly up-regulated the migration rate of A549 and HeLa cells.Conversely, in the same cells, silencing of endogenous Smad3 or treatment with Smad3 inhibitor, SIS3, down-regulated the migration rate .Conclusions Smad3 promotes cell migration of A549 and HeLa cells.
ABSTRACT
Inteleukin-37 is a member of IL-1 family and originally named as IL-1F7.It has five different subtypes (IL-37a-e).It has been reported that IL-37 suppresses pro-inflammatory cytokines production of a variety of immune cells, and further regulate innate immune response. In addition, IL-37 has protective effects on colitis, arthritis, pancreatitis and other inflammatory diseases that induced by exogenous stimuli. In a word, IL-37 is a novel anti-inflammatory cytokine and plays an important role in a variety of inflammation-related diseases.