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Objective To perfect the purification method of recombinant fusion protein of Hespintor (rHespintor) for increasing the protein extraction efficiency,and to investigate its effects on the proliferation,migration and invasion of hepatoblastoma cell line HepG2.Methods In the recombinant protein extraction,the inclusion body washing process was added and the protein purification buffer system was changed.BAPNA was used as the substrate.The inhibitory effect tof purified rHespintor on trypsin hydrolysis was detected.The blank group served as the control group.The MTT test,cell scratch wound healing test and tumor cell invasion test were performed to detect the effect of rHespintor on growth of hepatoblastoma HepG2 cells and its effect.Results The urea gradient washing on the inclusion body protein could effectively remove the vast majority of impure proteins from the targeted protein.After one-step purification,the target protein rHespintor exhibited a high inhibition effect of trypsin hydrolysis,and the inhibitory effect was exhibited a dose-dependent manner.After acting on hepatoblastoma HepG2 cells with rHespintor,the cell proliferation ability was inhibited,the migration ability was reduced and the number of invaded cells were significantly decreased.Conclusion rHespintor can significantly inhibit the proliferation,migration and invasion of hepatoblastoma cell line HepG2 cells in vitro.
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Objective To investigate the expression and significance of Maspin and IKKα in nasosinusoidal mucosa of rats with fungal rhinosinusitis (FRS).Methods A total of 40 SD rats were used to establish the FRS model, and randomly divided into nasal obstruction group, FRS group, immunosuppressive group and invasive FRS group, 10 rats in each group.Another 10 normal rats were used as control group.Mice in the control group were fed with normal diet.In the nasal obstruction group, the mice had only hemostatic cotton stuffed in the nasal cavity and injection of 0.9% NaCl in the abdominal and nasal cavities.In the FRS group, the mice were injected Aspergillus fumigatus spore suspension into the nasal cavity and 0.9% NaCl i.p.The mice of the immunosuppressive group were given cyclophosphamide i.p.and 0.9% NaCl injection into the nasal cavity.The invasive FRS group was injected with cyclophosphamide i.p.and Aspergillus fumigatus spore suspension into the nasal cavity.The serum levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were measured by enzyme-linked immunosorbent assay (ELISA).The expression of Maspin and IKKα in nasosinusoidal mucosa was detected by immunohistochemical staining.The expression of Maspin mRNA and IKKα mRNA in the nasosinusoidal mucosa was detected by fluorescence quantitative PCR.Results The serum levels of IL-6 and TNF-α in different groups were significantly different (P 0.05).Theresult of immunohistochemical staining showed that the protein expression of Maspin in the FRS group and invasive FRS group was significantly lower than that in the control group, nasal obstruction group and immunosuppressive group, while the expression of IKKα protein was significantly higher than that of control group, nasal obstruction group and immunosuppressive group (P< 0.05).The protein expression of Maspin in the invasive FRS group was significantly lower than that in the FRS group, by contrast, the expression of IKKα protein was significantly higher (P< 0.05).The PCRresult revealed that the expression levels of Maspin and IKKα mRNA were (0.217 ± 0.013) and (0.193 ± 0.012), significantly lower than that in the control, obstruction and immunosuppressive groups [(0.309 ± 0.021), (0.302 ± 0.017), and (0.293 ± 0.02)] (P< 0.05), while the expressions level of IKKα mRNA were significantly higher [(0.319 ± 0.043), (0.384 ± 0.048) vs (0.169 ± 0.015), (0.171 ± 0.018), and (0.175 ± 0.019)] (P< 0.05).Conclusions Down-regulation of Maspin expression after IKKα activation is the main cause of the onset of FRS, which may also be one of the mechanisms of invasive FRS.
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Objective To observe the expression of estrogen receptors (ERαand ERβ) on gastric cancer cells and evaluate the effect of Tamoxifen(TAM) on the cell proliferation and expression of serine proteinase inhibitor 9(PI9) of gastric cancer cells . Methods PI9 positive expression(MNK45 ,SGC7901)and negative expression (BGC823)of gastric cancer cell lines were from pre‐liminary screened ,the expression of ERα and ERβ detected by immunofluorescence chemical method ,the cell proliferation and ex‐pression of PI9 were tested by CCK8 assay and reverse transcription‐PCR after intervention of TAM .Results ERαprotein expres‐sion was noted in MNK45 and SGC7901 ,ERβwas noted in BGC823 ,but the expression of ERαand ERβwere not appear to be obvi‐ous after the intervention of TAM .Tamoxifen could obviously inhibited cell proliferation of MNK45 and SGC7901 at concentration of 0 .1-100 .0 μmol/L ,the differences were statistically significant compared with negative control group (P<0 .05) ,but showed no dose‐dependent to the proliferation of BGC823 and MNK28 .After treating with TAM ,the expression of PI9 mRNA of SGC7901 (0 .402± 0 .020) and MNK45(0 .359 ± 0 .048) were obviously lower than that in the negatwe control group(P< 0 .05). Conclusion Tamoxifen could significantly inhibit the proliferation of PI9 positive expression than PI9 negative expression of gastric cancer cell lines ,and showed obviously dose‐dependent ,its role in inhibiting proliferation might closely related to immune tolerance improved by PI9 .
ABSTRACT
Serine proteinase inhibitor9(P1-9),a charac-teristic member of serpins,has been identified as the only inhibitor of granzyme B(GrB).Accumulated evidence suggested that PI-9 inhibits GrB-induced apoptosis by blocking DNA fragmentation of target cell.Physiologically,PI-9 could protect cytotoxic lymphocytes from committing autolysis or fratricide,and play an important role in facilitating immunologic tolerance of immune-privileged sites.In addition,evidences in recent years suggest that PI-9 Was also involved in vailous pathologic processes,such as inflammation,trans plantation and immune tolerance of tumor.