AIM： To study the secretory expression of human hepatocyte growth factor （hdHGF） gene in Pichia pastoris. METHODS： The full-length gene of human cDNA encoding the deleted variant of hdHGF was cloned by RT-PCR and ...AIM： To study the secretory expression of human hepatocyte growth factor （hdHGF） gene in Pichia pastoris. METHODS： The full-length gene of human cDNA encoding the deleted variant of hdHGF was cloned by RT-PCR and overlapping-fragment PCR technique using mRNA of human placenta as a template. The cloned hdHGF cDNA was inserted into the Escherichia coilyeast shuttle vector of pPIC9. The constructed plasmid, pPIC9-hdHGF, was transformed into the GSl15 cells of the methylotrophic yeast, P pastoris, using a chemical method. The Hut ＋ transformants were screened to obtain high-expression strains by the test and analysis of expressed products of shake-flask culture. A secretory form of rhdHGF was made with the aid of the leader peptide sequence of Saccharomyces cerevisiae α-factor. RESULTS： The expressed products, which showed a band of molecular mass of about 80 ku, were observed on 15% SDS-PAGE and identified by Western blotting and N-terminal amino acid sequencing. In the high cell density culture of 5 L fermentor by fed-batch culture protocol, the cell biomass was reached at approximately 135 g （DCW）/L. The productivity of secreted total supernant protein concentration attained a high-level expression of more than 8.0 g/L and the ratio of rhdHGF band area was about 12.3% of the total band area scanned by SDS-PAGE analysis, which estimated that the product of rhdHGF was 500-900 mg/L.CONCLUSION： The P pastoris system represents an attractive tool of generating large quantities of hdHGF for both research and industrial purposes.展开更多
Objective:To investigate the changes of inflammatory factors, cellular immunity and humoral immunity in patients with secretory otitis media.Methods:A total of 82 cases of secretory otitis media admitted in our hospit...Objective:To investigate the changes of inflammatory factors, cellular immunity and humoral immunity in patients with secretory otitis media.Methods:A total of 82 cases of secretory otitis media admitted in our hospital from January 2017 to December 2017 were selected as the observation group, and 80 healthy volunteers in our hospital were selected as the control group. The tumor necrosis factor-α (TNF-α), calcitonin (PCT), platelet activating factor (PAF), endothelin-1 (ET-1), CD3+, CD4+, CD8+, CD4+/CD8+, IgA, IgG, IgM. were detected and compared.Results: The levels of TNF-α, PCT, PAF and ET-1 in the observation group were (2.21 ± 0.13) ng/mL, (3.96 ± 0.81) ng/mL, (149.50 ± 21.08) ng/mL, and (1.67 ± 0.53) μg/L, which were all higher than those of the control group, the differences were significant. The levels of CD3+, CD4+ and CD4+/CD8+ in the observation group were (51.95 ± 4.47)%, (37.04 ± 3.94)% and (1.10 ± 0.04) respectively, which were all lower than those in the control group, the differences were significant. The level of CD8+ in the observation group was (33.63 ± 3.94)%, higher than that in the control group, the difference was significant. The levels of IgA, IgG and IgM in the observation group were (4.97 ± 0.22) g/L, (31.16 ± 2.53) g/L and (5.12 ± 0.17) g/L respectively, which were all higher than the control group, the differences were significant.Conclusion:Inflammatory factors and immune status of patients with secretory otitis media are abnormal. It is suggested to strengthen clinical monitoring of relevant indicators.展开更多
Kazal-type serine protease inhibitor is one of the most important and widely distributed protease inhibitor families. Pancreatic secretory trypsin inhibitor (PSTI), also known as serine protease inhibitor Kazal type I...Kazal-type serine protease inhibitor is one of the most important and widely distributed protease inhibitor families. Pancreatic secretory trypsin inhibitor (PSTI), also known as serine protease inhibitor Kazal type I(SPINK1), binds rapidly to trypsin, inhibits its activity and is likely to protect the pancreas from prematurely activated trypsinogen. Therefore, it is an important factor in the onset of pancreatitis. Recent studies found that PSTI/SPINK1 is also involved in self-regulation of acinar cell phagocytosis, proliferation and growth of a variety of cell lines. In addition, it takes part in the response to inflammatory factor or injury and is highly related to adult type II citrullinemia.展开更多
Objective：To construct a Pichia pastoris （P. pastoris） expression vector of human intestinal trefoil factor （hITF） and study its expression and purification procedures. Methods：hITF gene encoding mature peptide ...Objective：To construct a Pichia pastoris （P. pastoris） expression vector of human intestinal trefoil factor （hITF） and study its expression and purification procedures. Methods：hITF gene encoding mature peptide was modified with a polybistidine tag sequence at the N-terminal, and then inserted into the P. pastoris expression vector pGAPZaA at the downstream of the s-mating factor signal. After gene sequencing, the recombinant pGAPZaA-hITF was transformed into the P. pastoris strain X-33 with lithium chloride, rhITF was induced to constitutively express in shake flask, and then analyzed with Tricine SDS-PAGEand Western blotting. The obtained rhITF was isolated from the cultured supernatants by ammonium sulfate precipitation, Ni-NTA affinity chromatography, and ultrafiltration. Results：The correctness and integrity of rhITF were identified by restriction digestion and gene sequencing, rhITF was successfully expressed to 50 mg/L as a secretive protein. After purification, the purity was above 95%. Tricine SDS-PAGE and Western-blot analysis showed that rhITF presented as a single band with a molecular weight of 10 kDa, a little larger than 7 879 Da as assayed by mass spectrometry analysis. Conclusion： hITF P. pastoris expression vector is successfully constructed and rhITF is expressed in P. pastoris at commercially relevant level. This research lays foundation for the further functional studying of hITF.展开更多
BACKGROUND The Chinese medicine Yangyin Huowei mixture(YYHWM)exhibits good clinical efficacy in the treatment of chronic atrophic gastritis(CAG),but the mechanisms underlying its activity remain unclear.AIM To investi...BACKGROUND The Chinese medicine Yangyin Huowei mixture(YYHWM)exhibits good clinical efficacy in the treatment of chronic atrophic gastritis(CAG),but the mechanisms underlying its activity remain unclear.AIM To investigate the therapeutic effects of YYHWM and its underlying mechanisms in a CAG rat model.METHODS Sprague-Dawley rats were allocated into control,model,vitacoenzyme,and low,medium,and high-dose YYHWM groups.CAG was induced in rats using Nmethyl-N′-nitro-N-nitrosoguanidine,ranitidine hydrochloride,hunger and satiety perturbation,and ethanol gavage.Following an 8-wk intervention period,stomach samples were taken,stained,and examined for histopathological changes.ELISA was utilized to quantify serum levels of PG-I,PG-II,G-17,IL-1β,IL-6,and TNF-α.Western blot analysis was performed to evaluate protein expression of IL-10,JAK1,and STAT3.RESULTS The model group showed gastric mucosal layer disruption and inflammatory cell infiltration.Compared with the blank control group,serum levels of PGI,PGII,and G-17 in the model group were significantly reduced(82.41±3.53 vs 38.52±1.71,23.06±0.96 vs 11.06±0.70,and 493.09±12.17 vs 225.52±17.44,P<0.01 for all),whereas those of IL-1β,IL-6,and TNF-αwere significantly increased(30.15±3.07 vs 80.98±4.47,69.05±12.72 vs 110.85±6.68,and 209.24±11.62 vs 313.37±36.77,P<0.01 for all),and the protein levels of IL-10,JAK1,and STAT3 were higher in gastric mucosal tissues(0.47±0.10 vs 1.11±0.09,0.49±0.05 vs 0.99±0.07,and 0.24±0.05 vs 1.04±0.14,P<0.01 for all).Compared with the model group,high-dose YYHWM treatment significantly improved the gastric mucosal tissue damage,increased the levels of PGI,PGII,and G-17(38.52±1.71 vs 50.41±3.53,11.06±0.70 vs 15.33±1.24,and 225.52±17.44 vs 329.22±29.11,P<0.01 for all),decreased the levels of IL-1β,IL-6,and TNF-α(80.98±4.47 vs 61.56±4.02,110.85±6.68 vs 89.20±8.48,and 313.37±36.77 vs 267.30±9.31,P<0.01 for all),and evidently decreased the protein levels of IL-10 and STAT3 in gastric mucosal tissues(1.11±0.09 vs 0.19±0.07 and 1.04±0.14 vs 0.55±0.09,P<0.01 for both).CONCLUSION YYHWM reduces the release of inflammatory factors by inhibiting the IL-10/JAK1/STAT3 pathway,alleviating gastric mucosal damage,and enhancing gastric secretory function,thereby ameliorating CAG development and cancer transformation.展开更多
基金Supported by the grants from National High Technology ResearchDevelopment Program, No. 2002AA2Z345BNo. 2004AA2Z3803 of the Ministry of Science and Technology of China
文摘AIM： To study the secretory expression of human hepatocyte growth factor （hdHGF） gene in Pichia pastoris. METHODS： The full-length gene of human cDNA encoding the deleted variant of hdHGF was cloned by RT-PCR and overlapping-fragment PCR technique using mRNA of human placenta as a template. The cloned hdHGF cDNA was inserted into the Escherichia coilyeast shuttle vector of pPIC9. The constructed plasmid, pPIC9-hdHGF, was transformed into the GSl15 cells of the methylotrophic yeast, P pastoris, using a chemical method. The Hut ＋ transformants were screened to obtain high-expression strains by the test and analysis of expressed products of shake-flask culture. A secretory form of rhdHGF was made with the aid of the leader peptide sequence of Saccharomyces cerevisiae α-factor. RESULTS： The expressed products, which showed a band of molecular mass of about 80 ku, were observed on 15% SDS-PAGE and identified by Western blotting and N-terminal amino acid sequencing. In the high cell density culture of 5 L fermentor by fed-batch culture protocol, the cell biomass was reached at approximately 135 g （DCW）/L. The productivity of secreted total supernant protein concentration attained a high-level expression of more than 8.0 g/L and the ratio of rhdHGF band area was about 12.3% of the total band area scanned by SDS-PAGE analysis, which estimated that the product of rhdHGF was 500-900 mg/L.CONCLUSION： The P pastoris system represents an attractive tool of generating large quantities of hdHGF for both research and industrial purposes.
文摘Objective:To investigate the changes of inflammatory factors, cellular immunity and humoral immunity in patients with secretory otitis media.Methods:A total of 82 cases of secretory otitis media admitted in our hospital from January 2017 to December 2017 were selected as the observation group, and 80 healthy volunteers in our hospital were selected as the control group. The tumor necrosis factor-α (TNF-α), calcitonin (PCT), platelet activating factor (PAF), endothelin-1 (ET-1), CD3+, CD4+, CD8+, CD4+/CD8+, IgA, IgG, IgM. were detected and compared.Results: The levels of TNF-α, PCT, PAF and ET-1 in the observation group were (2.21 ± 0.13) ng/mL, (3.96 ± 0.81) ng/mL, (149.50 ± 21.08) ng/mL, and (1.67 ± 0.53) μg/L, which were all higher than those of the control group, the differences were significant. The levels of CD3+, CD4+ and CD4+/CD8+ in the observation group were (51.95 ± 4.47)%, (37.04 ± 3.94)% and (1.10 ± 0.04) respectively, which were all lower than those in the control group, the differences were significant. The level of CD8+ in the observation group was (33.63 ± 3.94)%, higher than that in the control group, the difference was significant. The levels of IgA, IgG and IgM in the observation group were (4.97 ± 0.22) g/L, (31.16 ± 2.53) g/L and (5.12 ± 0.17) g/L respectively, which were all higher than the control group, the differences were significant.Conclusion:Inflammatory factors and immune status of patients with secretory otitis media are abnormal. It is suggested to strengthen clinical monitoring of relevant indicators.
基金Supported by the National Basic Research 973 Pre-research Program of China,No.2006CB708506
文摘Kazal-type serine protease inhibitor is one of the most important and widely distributed protease inhibitor families. Pancreatic secretory trypsin inhibitor (PSTI), also known as serine protease inhibitor Kazal type I(SPINK1), binds rapidly to trypsin, inhibits its activity and is likely to protect the pancreas from prematurely activated trypsinogen. Therefore, it is an important factor in the onset of pancreatitis. Recent studies found that PSTI/SPINK1 is also involved in self-regulation of acinar cell phagocytosis, proliferation and growth of a variety of cell lines. In addition, it takes part in the response to inflammatory factor or injury and is highly related to adult type II citrullinemia.
基金Supported by the National Natural Science Foundation of China (No. 30200294)
文摘Objective：To construct a Pichia pastoris （P. pastoris） expression vector of human intestinal trefoil factor （hITF） and study its expression and purification procedures. Methods：hITF gene encoding mature peptide was modified with a polybistidine tag sequence at the N-terminal, and then inserted into the P. pastoris expression vector pGAPZaA at the downstream of the s-mating factor signal. After gene sequencing, the recombinant pGAPZaA-hITF was transformed into the P. pastoris strain X-33 with lithium chloride, rhITF was induced to constitutively express in shake flask, and then analyzed with Tricine SDS-PAGEand Western blotting. The obtained rhITF was isolated from the cultured supernatants by ammonium sulfate precipitation, Ni-NTA affinity chromatography, and ultrafiltration. Results：The correctness and integrity of rhITF were identified by restriction digestion and gene sequencing, rhITF was successfully expressed to 50 mg/L as a secretive protein. After purification, the purity was above 95%. Tricine SDS-PAGE and Western-blot analysis showed that rhITF presented as a single band with a molecular weight of 10 kDa, a little larger than 7 879 Da as assayed by mass spectrometry analysis. Conclusion： hITF P. pastoris expression vector is successfully constructed and rhITF is expressed in P. pastoris at commercially relevant level. This research lays foundation for the further functional studying of hITF.
基金Supported by the Project of Regional Collaborative Innovation of Xinjiang Uygur Autonomous Region,No.2022E01008the Graduate Innovation Project of Xinjiang Medical University,No.CXCY2023012.
文摘BACKGROUND The Chinese medicine Yangyin Huowei mixture(YYHWM)exhibits good clinical efficacy in the treatment of chronic atrophic gastritis(CAG),but the mechanisms underlying its activity remain unclear.AIM To investigate the therapeutic effects of YYHWM and its underlying mechanisms in a CAG rat model.METHODS Sprague-Dawley rats were allocated into control,model,vitacoenzyme,and low,medium,and high-dose YYHWM groups.CAG was induced in rats using Nmethyl-N′-nitro-N-nitrosoguanidine,ranitidine hydrochloride,hunger and satiety perturbation,and ethanol gavage.Following an 8-wk intervention period,stomach samples were taken,stained,and examined for histopathological changes.ELISA was utilized to quantify serum levels of PG-I,PG-II,G-17,IL-1β,IL-6,and TNF-α.Western blot analysis was performed to evaluate protein expression of IL-10,JAK1,and STAT3.RESULTS The model group showed gastric mucosal layer disruption and inflammatory cell infiltration.Compared with the blank control group,serum levels of PGI,PGII,and G-17 in the model group were significantly reduced(82.41±3.53 vs 38.52±1.71,23.06±0.96 vs 11.06±0.70,and 493.09±12.17 vs 225.52±17.44,P<0.01 for all),whereas those of IL-1β,IL-6,and TNF-αwere significantly increased(30.15±3.07 vs 80.98±4.47,69.05±12.72 vs 110.85±6.68,and 209.24±11.62 vs 313.37±36.77,P<0.01 for all),and the protein levels of IL-10,JAK1,and STAT3 were higher in gastric mucosal tissues(0.47±0.10 vs 1.11±0.09,0.49±0.05 vs 0.99±0.07,and 0.24±0.05 vs 1.04±0.14,P<0.01 for all).Compared with the model group,high-dose YYHWM treatment significantly improved the gastric mucosal tissue damage,increased the levels of PGI,PGII,and G-17(38.52±1.71 vs 50.41±3.53,11.06±0.70 vs 15.33±1.24,and 225.52±17.44 vs 329.22±29.11,P<0.01 for all),decreased the levels of IL-1β,IL-6,and TNF-α(80.98±4.47 vs 61.56±4.02,110.85±6.68 vs 89.20±8.48,and 313.37±36.77 vs 267.30±9.31,P<0.01 for all),and evidently decreased the protein levels of IL-10 and STAT3 in gastric mucosal tissues(1.11±0.09 vs 0.19±0.07 and 1.04±0.14 vs 0.55±0.09,P<0.01 for both).CONCLUSION YYHWM reduces the release of inflammatory factors by inhibiting the IL-10/JAK1/STAT3 pathway,alleviating gastric mucosal damage,and enhancing gastric secretory function,thereby ameliorating CAG development and cancer transformation.
