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1.
Chinese Journal of Preventive Medicine ; (12): 659-667, 2022.
Artículo en Chino | WPRIM | ID: wpr-935340

RESUMEN

Coronavirus disease 19 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 has spread all over the world. Streptococcus pneumoniae as a common pathogen of community-acquired pneumonia shares similar high-risk susceptible populations with COVID-19. Streptococcus pneumoniae co-infection is a key risk factor for severe COVID-19 and death. Pneumococcal vaccination has a beneficial impact on reducing the incidence and mortality of COVID-19. The vaccination rate of streptococcus pneumoniae is still low in China. Streptococcus pneumoniae vaccination may be one of effective strategies in the management of COVID-19 for high-risk population such as the elderly and those who have underlying chronic diseases.


Asunto(s)
Anciano , Humanos , COVID-19 , Coinfección , Infecciones Neumocócicas/prevención & control , Streptococcus pneumoniae , Vacunación
2.
China Journal of Chinese Materia Medica ; (24): 1166-1172, 2015.
Artículo en Chino | WPRIM | ID: wpr-246131

RESUMEN

To study the effect of Fuzheng Huayu recipe (FZHY) on five types of isozymes of cytochrome P450 (CYP450) of normal and liver fibrosis rats by using the cocktail probe method. Dimethylnitrosamine ( DMN) was injected to induce the liver fibrosis model. After the tail vein injection with Cocktail probe solutions prepared with five CYP450s probe substrates (phenacetin-CYP1A2, omeprazole-CYP2C9, tolbutamide-CYP2C19, dextromethorphan-CYP2D6, midazolam-CYP3A4), the plasma concentrations of the five probe substrates were determined by LC-MS/MS, and the pharmacokinetic parameters were calculated by PK solutions 2. After the oral administration with FZHY, normal rats given phenacetin, omeprazole, tolbutamide and dextromethorphan showed increase in AUC(0-t) and decrease in CL to varying degrees, indicating that FZHY obviously inhibited the activities of CYP1A2, CYP2C9, CYP2C19 and CYP2D6 in normal rats, but with no obvious effect on the activity of CYP3A4. After the oral administration with FZHY, liver fibrosis rats treated with CYP2C9 showed the significant increase in AUC(0-t) and significant decrease in Vd, hut with no obvious changes in the pharmacokinetic parameters of other four types of prove substances, suggesting that FZHY could significantly inhibit the activity of CYP2C9 in rats but had no effect on the activities of CYP1A2, CYP2C19, CYP2D6 and CYP3A4. The changes in the activity of CYP450 isozymes in liver fibrosis rats may be the reason for FZHY's different effects on CYP450 isozymes in normal and liver fibrosis rats.


Asunto(s)
Animales , Humanos , Masculino , Ratas , Sistema Enzimático del Citocromo P-450 , Genética , Metabolismo , Modelos Animales de Enfermedad , Medicamentos Herbarios Chinos , Química , Farmacocinética , Isoenzimas , Genética , Metabolismo , Cirrosis Hepática , Quimioterapia , Genética , Espectrometría de Masas , Ratas Wistar
3.
Biomedical and Environmental Sciences ; (12): 273-278, 2010.
Artículo en Inglés | WPRIM | ID: wpr-360592

RESUMEN

<p><b>OBJECTIVE</b>Cytotoxin-associated protein (CagA) of H. pylori has been confirmed to be closely associated with gastric inflammation and tumorigenesis, but the mechanism behind it is little understood. In this study, we try to determine roles of CagA(+) strain in activating PI3K/Akt1 signaling pathway, and affecting expression of p21(WAF1/CIP1) and p27(KIP1), and also in releasing IL-8 in host cells.</p><p><b>METHODS</b>Akt1 phosphorylation and IL-8 levels of CagA(+) and CagA⁻ strain infected AGS cells were detected by ELISAs. Two quantitative RT-PCRs were established to measure p21(WAF1/CIP1) and p27(KIP1) mRNA levels in the CagA(+) and CagA⁻ strain infected cells. LY294002, an inhibitor of PI3K/Akt pathway, was used to define effect of the pathway in IL-8 release.</p><p><b>RESULTS</b>CagA(+) strain could induce an obvious elevation of Akt1 phosphorylation in the infected AGS cells while CagA? strain failed to do so. The CagA(+) H. pylori strain infected AGS cells showed significant drops both in p21(WAF1/CIP1) and p27(KIP1) mRNA levels, whereas the CagA⁻ H. pylori strain caused a remarkable increase in p21(WAF1/CIP1) mRNA without affecting p27(KIP1) gene transcription in the AGS cells. Both the CagA(+) and CagA⁻ H. pylori strains enabled AGS cells to produce close elevated levels of IL-8, and the LY294002 block resulted in unexpected elevations of IL-8 levels.</p><p><b>CONCLUSIONS</b>CagA can activate PI3K/Akt1 pathway that plays an inhibitory role in IL-8 release in H. pylori infected AGS cells. Activation of PI3K/Akt1 pathway and subsequent negative regulation of p21(WAF1/CIP1) and p27(KIP1) expression might be involved in CagA-associated carcinogenesis.</p>


Asunto(s)
Humanos , Antígenos Bacterianos , Genética , Fisiología , Proteínas Bacterianas , Genética , Fisiología , Línea Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Mucosa Gástrica , Biología Celular , Microbiología , Helicobacter pylori , Metabolismo , Virulencia , Fisiología , Interleucina-8 , Secreciones Corporales , Péptidos y Proteínas de Señalización Intracelular , Metabolismo , Fosfatidilinositol 3-Quinasas , Metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt , Metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Transcripción Genética , Virulencia
4.
Journal of Zhejiang University. Medical sciences ; (6): 364-372, 2008.
Artículo en Chino | WPRIM | ID: wpr-344320

RESUMEN

<p><b>OBJECTIVE</b>To determine the PD-L1 expression levels in circulating dendritic cells(DCs) of patients with HBeAg positive chronic hepatitis B, and to investigate the effects of anti-PD-L1 antibody on DCs stimulating capacity of allogeneic lymphocytes.</p><p><b>METHODS</b>DCs were separated and induced from 22 HBeAg positive chronic hepatitis B patients (CHB), 8 acute resolved hepatitis B patients (AHB) and 10 healthy blood donors. PD-L1 and PD-L2 expression in DCs were determined using real-time RT-PCR and flow cytometry. The potential of circulating DCs on the proliferation of allogeneic T cells was detected and a specific monoclonal antibody against PD-L1 was used in alternative experiments. Serum HBV-DNA titers were measured using real-time PCR, and HBV markers and liver function were also evaluated.</p><p><b>RESULT</b>The expression of PD-L1 but not PD-L2 was upregulated in circulating DCs of CHB patients, compared to AHB patients and healthy controls (both P<0.01). CHB patients with greater than 106 copies /ml of serum HBV DNA loads had a higher level of PD-L1 in circulating DCs than those with less than 106 copies/ml (P<0.05), and the high expression of PD-L1 in DCs was positively correlated with the plasma viral load. Moreover, the potential of circulating DCs from CHB patients was significantly decreased compared with healthy controls or AHB patients, while the blockade of PD-L1 using anti-PD-L1 monoclonal antibody increased the ability of DCs on the proliferation of allogeneic T cells in vitro.</p><p><b>CONCLUSION</b>High expression of PD-L1 on circulating DCs may be associated with T cell exhaustion and persistent high levels of HBV DNA replication in chronic hepatitis B patients.</p>


Asunto(s)
Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Adulto Joven , Antígenos CD , Metabolismo , Proteínas Reguladoras de la Apoptosis , Metabolismo , Células Dendríticas , Alergia e Inmunología , Metabolismo , Antígenos e de la Hepatitis B , Sangre , Hepatitis B Crónica , Alergia e Inmunología , Metabolismo , Patología , Receptor de Muerte Celular Programada 1 , ARN Mensajero , Metabolismo , Linfocitos T , Alergia e Inmunología
5.
Chinese Journal of Applied Physiology ; (6): 468-473, 2008.
Artículo en Chino | WPRIM | ID: wpr-252738

RESUMEN

<p><b>AIM</b>To analyse the alterations of protein expression in the kidney of spontaneously hypertensive rat with losartan.</p><p><b>METHODS</b>The proteins of the kidney were isolated by two-dimensional gel electrophoresis. The protein spots with significant changes were selected for further identification by LC-MS/MS.</p><p><b>RESULTS</b>The number of average protein spots of two groups was 570 +/- 48 and 686 +/- 30 respectively. Compared with the SHR, 13 spots had changed significantly after treated with losartan. There were 5 protein spots detected only in SHR group, while 4 up-regulated and 4 down-regulated protein spots were detected in SHR-L group. These differentially expressed proteins were detected by mass spectrometry. 7 spots were identified. There were Heat shock protein (Hsp), Tubulin alpha-1 chain, Transthyretin precursor, Liver regeneration-related protein LRRG03, Ezrin-radixin-moesin binding phosphoprotein 50, Phosphoglycerate kinase 1 and Anionic trypsin I precursor.</p><p><b>CONCLUSION</b>The different protein spots expression may play important roles in Losartan's effective protection to hypertension rats renal tissue.</p>


Asunto(s)
Animales , Masculino , Ratas , Bloqueadores del Receptor Tipo 1 de Angiotensina II , Farmacología , Usos Terapéuticos , Cromatografía Liquida , Electroforesis en Gel Bidimensional , Métodos , Hipertensión , Quimioterapia , Metabolismo , Riñón , Metabolismo , Losartán , Farmacología , Usos Terapéuticos , Proteoma , Metabolismo , Proteómica , Métodos , Ratas Endogámicas SHR , Espectrometría de Masas en Tándem
6.
Journal of Zhejiang University. Medical sciences ; (6): 543-548, 2007.
Artículo en Chino | WPRIM | ID: wpr-344401

RESUMEN

<p><b>OBJECTIVE</b>To investigate the anti-fibrosis mechanism of radix astragali through the observation on regulating cytokine secretion by astragalosides and astragalus polysaccharides in hepatic stellate cell line LX-2.</p><p><b>METHODS</b>Astragalus polysaccharides and astragalosides at concentration of 25 microg/ml and 300 microg/ml were added to LX-2 cell culture.After 24 h culture total RNA contents were extracted and TGF-beta1, HGF, MP2 and MMP9 mRNA were measured by real time fluorescence quantification PCR technique. The cell growth curves were detected by Real Time Cell Electronics Sensing.</p><p><b>RESULTS</b>300 microg/ml of astragalus polysaccharides suppressed LX-2 cell proliferation (P<0.05, in 94.7 h), while astragalosides induced an significant cell proliferation (P<0.05) both at lower and higher concentration. 25 microg/ml astragalus polysaccharides and astragalosides induced TGF-beta1 expression. For other cytokines, astragalus polysaccharides induced a 104.9 fold higher expression of MMP2, while astragalosides induced a 550.65 fold higher expression of IL-10. Astragalus polysaccharides at 300 microg/ml concentration exhibited an inhibition effect on TGF-beta1, HGF, MMP9 and IL-10 mRNA expression, while up-regulated MMP2 mRNA expression. Astragalosides at 300 mug/ml concentration inhibited TGF-beta1 mRNA expression, while up-regulated MMP2, MMP9 and IL-10 mRNA expression.</p><p><b>CONCLUSION</b>The anti-fibrosis function of astragalus polysaccharides might be associated with up-regulation of MMP2 expression, while that of astragalosides with up-regulation of MMP2, MMP9 and IL-10 expression.</p>


Asunto(s)
Humanos , Astragalus propinquus , Química , Línea Celular , Citocinas , Secreciones Corporales , Células Estrelladas Hepáticas , Biología Celular , Metabolismo , Interleucina-10 , Genética , Metabolismo , Metaloproteinasa 2 de la Matriz , Genética , Metabolismo , Metaloproteinasa 9 de la Matriz , Genética , Metabolismo , Raíces de Plantas , Química , Polisacáridos , Farmacología , ARN Mensajero , Genética , Metabolismo , Saponinas , Farmacología , Triterpenos , Farmacología , Regulación hacia Arriba
7.
Chinese Journal of Applied Physiology ; (6): 75-79, 2006.
Artículo en Chino | WPRIM | ID: wpr-254601

RESUMEN

<p><b>AIM</b>To explore the relationship between expression of signal transduction and activators of transcription 3 (STAT3) gene and proliferation of rat pulmonary arterial smooth muscle cells (PASMCs) under hypoxia conditions.</p><p><b>METHODS</b>After primarily cultured rat PASMCs was treated with AG490 and then exposed to hypoxia, the tyrosine-phosphorylated STAT3 protein were detected at 2 h, 6 h, 12 h, 16 h, 24 h of exposure to hypoxia by semi-quantitive RT-PCR (sqRT-PCR) and Western blot respectively. The expression of c-myc mRNA was analyzed by sqRT-PCR. 3H-TdR incorporation was used to detect the cell proliferation.</p><p><b>RESULTS</b>The level of tyrosine-phosphorylated STAT3 increased at 6 h and peaked at 12 h. The expression of c-myc mRNA increased after 2 h of hypoxia and reached maximal level at 4 h, then declined at 6 h and to the basal levels at 12 h. With the prolonging of hypoxia time, 3H-TdR incorporation in PASMC under hypoxia conditions was significantly higher. AG490 inhibited proliferation of PASMCs by preventing STAT3 tyrosine phosphorylation and the expression of c-myc under hypoxia conditions.</p><p><b>CONCLUSION</b>(1) The activation of STAT3 and c-myc gene might play an important role in the early stage of hypoxia-induced PASMCs proliferation. (2) STAT3 upregulated the expression of c-myc during the proliferation of PASMCs induced by hypoxia.</p>


Asunto(s)
Animales , Ratas , Hipoxia de la Célula , Proliferación Celular , Células Cultivadas , Hipoxia , Metabolismo , Músculo Liso Vascular , Biología Celular , Metabolismo , Miocitos del Músculo Liso , Metabolismo , Arteria Pulmonar , Biología Celular , Ratas Wistar , Factor de Transcripción STAT3 , Metabolismo , Transducción de Señal
8.
National Journal of Andrology ; (12): 25-27, 2006.
Artículo en Chino | WPRIM | ID: wpr-338376

RESUMEN

<p><b>OBJECTIVE</b>To investigate the relationship between four cytokines in serum and prostatic fluid and chronic abacterial prostatitis (CAP).</p><p><b>METHODS</b>The levels of interleukin-2 (IL-2), IL-4, IL-8, tumor necrosis factor-alpha (TNF-alpha) were detected in 86 patients with CAP [CAP I (n = 60), WBC > or = 10/HP; CAP II (n = 26, WBC < 10/HP)], and 20 healthy males as control by ELISA.</p><p><b>RESULTS</b>IL-2, IL-8 and TNF-alpha in serum and prostatic fluid, and IL-4 in serum in patients with CAP I were markedly higher than those in the control, respectively (P < 0.05 or P < 0.01). No significant difference was observed in IL-4 level in prostatic fluid among CAP I, CAP II and control groups respectively (P > 0.05), and in IL-2, IL4, TNF-alpha in serum and prostatic fluid between CAP II group and the control (P > 0.05). IL-8 was correlated with WBC number in prostatic fluid (r = 0.62, P < 0.05).</p><p><b>CONCLUSION</b>IL-2, IL-8, TNF-alpha may play roles in the process of pathogenesis of CAP, and they have applicable value in the diagnosis and typing of CAP.</p>


Asunto(s)
Adulto , Humanos , Masculino , Persona de Mediana Edad , Secreciones Corporales , Química , Enfermedad Crónica , Citocinas , Metabolismo , Ensayo de Inmunoadsorción Enzimática , Interleucina-2 , Metabolismo , Interleucina-4 , Metabolismo , Interleucina-8 , Metabolismo , Próstata , Secreciones Corporales , Prostatitis , Sangre , Metabolismo , Factor de Necrosis Tumoral alfa , Metabolismo
9.
Journal of Zhejiang University. Medical sciences ; (6): 4-8, 2005.
Artículo en Chino | WPRIM | ID: wpr-353258

RESUMEN

<p><b>OBJECTIVE</b>To determine the ability of adhering and invading to host cells and the related pathologic changes of Leptospira spp. with different virulence.</p><p><b>METHODS</b>A special Fontana silver staining method was developed to observe the ability of L.interrogans serogroup Icterohaemorrhagiae serovar lai strain 56601 and serogroup Pomona serovar pomona strain 56608, and L.biflexa serogroup Samaranga serovar patoc strain Patoc I to adhere Vero and J774A.1 cells. Ultrastructural lesions of the infected cells were examined by electron microscopy. By using flow cytometry with fluorescein labeling of FITC-Annexin V/PI,apoptosis and necrosis of the Vero and J774A.1 cells induced by the leptospiral strains before and after inactivation with ultraviolet treatment were detected, respectively.</p><p><b>RESULTS</b>The adhering rates of L.interrogans strains 56601 and 56608 to the Vero cells were 24.2% and 22.9% (P>0.05), while to the J774A.1 cells were 49.0% and 46.9% (P>0.05), respectively. L.biflexa strain Patoc I did not adhere to these two host cells. After the two strains of L.interrogans invaded two different cell lines, the special phagosomes containing the Leptospira were formed and similar cell ultrastructural lesions, such as chromatilysis and chromatin condensation, vacuolar degeneration, mitochondrion swelling and mitochondrial crista disappearance, and endoplasmic reticulum swelling and paramembranous ribosome disappearance, were observed. The apoptosis rates in the Vero cells caused by the L.interrogans strains 56601 and 56608 before and after ultraviolet inactivation were 84.4%, 82.8% and 77.9%, 86.1%, respectively. The L.interrogans strain 56601 mainly induced the terminal apoptosis in Vero cells with the rates of 68.0% and 52.9% before or after inactivation, while the L.interrogans strain 56608 mainly induced the early apoptosis in Vero cells with apoptosis rates of 64.1% and 50.1% before or after inactivation. In the J774A.1 cells, the L.interrogans strain 56608 caused cell necrosis (before and after inactivation) and apoptosis that was dominated by terminal apoptosis.</p><p><b>CONCLUSION</b>The established Fontana silver staining method can be used to observe adhesion of L.interrogans.L.interrogans can invade host cells through endocytosis which causes untrastructural lesions. The necrosis or apoptosis induced by L.interrogans are affected by different host cell lines but not the virulence of L.interrogans strains.</p>


Asunto(s)
Animales , Humanos , Apoptosis , Fisiología , Adhesión Celular , Células Cultivadas , Chlorocebus aethiops , Endocitosis , Leptospira interrogans , Clasificación , Virulencia , Macrófagos , Microbiología , Serotipificación , Células Vero , Virulencia
10.
Journal of Zhejiang University. Medical sciences ; (6): 15-20, 2005.
Artículo en Chino | WPRIM | ID: wpr-353256

RESUMEN

<p><b>OBJECTIVE</b>To determine the effects of leptospiral strains with different virulence on intracellular free Ca(2+)level and its relation with phospholipase C (PLC) activity of L.interrogans.</p><p><b>METHODS</b>L.interrogans-j infection cell modals were established with Vero and J774A.1 cell lines. Vero and J774A.1 cells were co-incubated with L.interrogans serogroup Icterohaemorrhagiae serovar lai strain 56601 (strong virulence) and serogroup Pomona serovar pomona strain 56608 (weak virulence) and L.biflexa serogroup Samaranga serovar patoc strain Patoc I (non-virulence). Intracellular free Ca(2+)levels were detected by laser scanning confocal microscopy with specific fluorescence labeling of fluoj3/AM. Using [(3)H] PIP2 as the substrate, the PLC activities in the culture supernatant, and cytoplasma and cytomembrane of the three strains of Leptospira were measured by isotope assay.</p><p><b>RESULTS</b>The baseline intracellular free Ca(2+)levels in the normal Vero and J774A.1 cells were (102.3+/-8.2)% and (105.9+/-7.3)%,respectively. The fluorescence intensity in the two cell lines incubated with L.biflexa strain Patoc I were fluctuated in range of (102.3+/-8.2)%approximate, equals(102.2+/-8.3)% during the observation period. The intracellular free Ca(2+)levels in the two cell lines infected with L.interrogans strain 56601 showed elevation with double peak patterns, with first peaks of (430.5+/-35.7)%, (747.5+/-18.5)% and the second peak of (380.6+/-17.4)%, (804.6+/-22.4)%, respectively. When the cells were infected with L.interrogans strain 56608, the intracellular free Ca(2+)levels were rising slowly with a single slope-like pattern, with the maximal of (235.0+/-19.3)% in Vero cells and (402.4+/-17.4)% in J774A.1 cells, which were significantly lower than those in the cells infected with L.interrogans strain 56601 (P<0.01). The culture supernatants, and cytoplasma and cytomembrane proteins of all three strains displayed PLC activities (P<0.05).</p><p><b>CONCLUSION</b>The cells infected with L.interrogans of different virulence show distinct intracellular free Ca(2+)levels and peak patterns. The different host cell lines can affect the intracellular free Ca(2+)levels, which is not related to the PLC activity in the leptospiral strains.</p>


Asunto(s)
Animales , Humanos , Calcio , Metabolismo , Células Cultivadas , Chlorocebus aethiops , Endocitosis , Leptospira interrogans , Virulencia , Macrófagos , Metabolismo , Microbiología , Fosfolipasas de Tipo C , Metabolismo , Células Vero , Virulencia
11.
Journal of Zhejiang University. Medical sciences ; (6): 21-26, 2005.
Artículo en Chino | WPRIM | ID: wpr-353255

RESUMEN

<p><b>OBJECTIVE</b>To construct prokaryotic expression systems of ltB/ctB-ompL1/1 fusion genes and to determine the L.interrogans carrying status in leptospirosis patients with the expression products.</p><p><b>METHODS</b>The fusion genes ltB-ompL1/1 and ctB-ompL1/1 were constructed using linking primer PCR method. SDS-PAGE was used to examine expression of the target recombinant proteins rLTB-rOmpL1/1 and rCTB-rOmpL1/1. Western blot and GM1-ELISA were used to measure the immunogenic and GM(1)-binding activities of rLTB-rOmpL1/1 and rCTB-rOmpL1/1, respectively. PCR and MAT were performed to detect the expression of ompL1 gene in 97 wild L.interrogans strains. Antibodies against ompL1 gene products in serum samples of 228 leptospirosis patients were detected with ELISA method.</p><p><b>RESULTS</b>The homogeneity of nucleotide and putative amino acid sequence of ltB-jompL1/1 and ctB-ompL1/1 fusion genes were 99.7 % - 99.9 % and 99.5 % - 100 %, in comparison with the reported corresponding sequences. Expression outputs of both rLTB-rOmpL1/1 and rCTB-rOmpL1/1, mainly present in inclusion body, accounted for 10% of the total bacterial protein. Both rLTB-rOmpL1/1 and rCTB-rOmpL1/1 could combine to rabbit anti-rOmpL1/1 serum and bovine GM(1). 89.7 % of L.interrogans wild strains had ompL1 gene. 87.6% of the wild L.interrogans strains presented positive results for MAT (titers :1:4-1:256) with the rabbit anti-rOmpL1/1 or anti-rOmpL1/2 sera. 86.8% and 88.6% of the patients' serum samples were positive for rOmpL1/1 and rOmpL1/2 antibodies, respectively.</p><p><b>CONCLUSION</b>The fusion proteins, rLTB-rOmpL1/1 and rCTB-rOmpL1/1, showed high immunogenic and GM(1)-binding activities. ompL1 gene is extensively distributed and frequently expressed in different serogroups of L.interrogans and its products expressed by different genotypes exhibit extensive cross-antigenicity.</p>


Asunto(s)
Humanos , Proteínas de la Membrana Bacteriana Externa , Genética , Alergia e Inmunología , Toxinas Bacterianas , Genética , Vacunas Bacterianas , Genética , Clonación Molecular , Enterotoxinas , Genética , Proteínas de Escherichia coli , Genética , Genes Bacterianos , Genética , Leptospira interrogans , Genética , Alergia e Inmunología , Células Procariotas , Metabolismo , Proteínas Recombinantes de Fusión , Genética , Alergia e Inmunología , Vacunas Sintéticas , Genética
12.
Journal of Zhejiang University. Medical sciences ; (6): 27-32, 2005.
Artículo en Chino | WPRIM | ID: wpr-353254

RESUMEN

<p><b>OBJECTIVE</b>To construct lipL32/1-lipL41/1 fusion gene and its prokaryotic expression system and to determine frequencies of carrying and expression of lipL32 and lipL41 genes in L.interrogans wild strains and specific antibody levels in sera from leptospirosis patients.</p><p><b>METHODS</b>lipL32/1-lipL41/1 fusion gene was constructed using linking primer PCR method and the prokaryotic expression system of the fusion gene done with routine techniques. SDS-PAGE was used to examine expression of the target recombinant protein rLipL32/1-rLipL41/1. Immunogenicity of rLipL32/1-rLipL41/1 was identified by Western blot. PCR and MAT were performed to detect carrying and expression of lipL32 and lipL41 genes in 97 wild L.interrogans strains. Antibodies against products of lipL32 and lipL41 genes in serum samples from 228 leptospirosis patients were detected by ELISA method.</p><p><b>RESULTS</b>The homogeneity of nucleotide and putative amino acid sequence of lipL32/1-lipL41/1 fusion gene were 99.9 % and 99.8 % in comparison with the reported sequences. Expression output of the target recombinant protein rLipL32/1-rLipL41/1, mainly present in inclusion body, accounted for 10 % of the total bacterial proteins. Both the rabbit antisera against rLipL32/1 and rLipL41/1 could combine to rLipL32/1-rLipL41/1. 97.9 % and 87.6 % of the L.interrogans wild strains had lipL32 and lipL41 genes, respectively. 95.9 % and 84.5 % of the wild strains were positive for MAT with titers of 1:4 - 1:128 using rabbit anti-rLipL32s or anti-rLipL41s sera, respectively. 94.7 % - 97.4 % of the patients'serum samples were positive for rLipL32s antibodies, while 78.5 % - 84.6 % of them were rLipL41s antibodies detectable.</p><p><b>CONCLUSION</b>lipL32/1-jlipL41/1 fusion gene and its prokaryotic expression system were successfully constructed. The expressed fusion protein had qualified immunogenicity. Both the lipL32 and lipL41 genes are extensively carried and frequently expressed by different serogroups of L.interrogans, and their expression products exhibit cross-antigenicity.</p>


Asunto(s)
Humanos , Anticuerpos Antibacterianos , Sangre , Antígenos Bacterianos , Genética , Alergia e Inmunología , Proteínas de la Membrana Bacteriana Externa , Genética , Clonación Molecular , Ensayo de Inmunoadsorción Enzimática , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Genética , Leptospira interrogans , Genética , Leptospirosis , Alergia e Inmunología , Microbiología , Lipoproteínas , Genética , Células Procariotas , Metabolismo , Proteínas Recombinantes de Fusión , Genética , Alergia e Inmunología
13.
Journal of Zhejiang University. Medical sciences ; (6): 33-42, 2005.
Artículo en Chino | WPRIM | ID: wpr-353253

RESUMEN

<p><b>OBJECTIVE</b>To construct the eukaryotic expression system of L.interrogans lipL32/1-ompL1/1 fusion gene and to identify the immunoreactivity of expression products.</p><p><b>METHODS</b>PCR with linking primer was used to construct the fusion gene lipL32/1-ompL1/1. The P.pastoris eukaryotic expression system of the fusion gene, pPIC9K-lipL32/1-ompL1/1-P. pastorisGS115, was constructed after the fusion gene was cloned and sequenced. Colony with phenotype His(+)Mut(+) was isolated by using MD and MM plates and His(+) Mut(+) transformant with high resistance to G418 was screened out by using YPD plate. Using lysate of His(+) Mut(+) colony with high copies of the target gene digested with yeast lyase as the template and 5'AOX1 and 3'AOX1 as the primers, the target fusion gene in chromosome DNA of the constructed P. pastoris engineering strain was detected by PCR. Methanol in BMMY medium was used to induce the target recombinant protein rLipL32/1-rOmpL1/1 expression. rLipL32/1-rOmpL1/1 in the medium supernatant was extracted by using ammonium sulfate precipitation and Ni-NTA affinity chromatography. Output and immunoreactivity of rLipL32/1-rOmpL1/1 were measured by SDS-PAGE and Western blot methods, respectively.</p><p><b>RESULTS</b>Amplification fragments of the obtained fusion gene lipL32/1-ompL1/1 was 1794 bp in size. The homogeneity of nucleotide and putative amino acid sequences of the fusion gene were as high as 99.94 % and 100 %, respectively, compared with the sequences of original lipL32/1 and ompL1/1 genotypes. The constructed eukaryotic expression system was able to secrete rLipL32/1-rOmpL1/1 with an output of 10 % of the total proteins in the supernatant, which located the expected position after SDS-PAGE. The rabbit anti-rLipL32/1 and anti-rOmpL1/1 sera could combine the expressed rLipL32/1-rOmpL1/1.</p><p><b>CONCLUSION</b>An eukaryotic expression system with high efficiency in P.pastoris of L.interrogans lipL32/1-ompL1/1 fusion gene was successfully constructed in this study. The expressed fusion protein shows specific immunoreactivity, which can be used as a potential antigen for developing a novel vaccine of L.interrogans.</p>


Asunto(s)
Humanos , Secuencia de Aminoácidos , Antígenos Bacterianos , Genética , Alergia e Inmunología , Proteínas de la Membrana Bacteriana Externa , Genética , Secuencia de Bases , Clonación Molecular , Células Eucariotas , Metabolismo , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Genética , Leptospira interrogans , Genética , Leptospirosis , Alergia e Inmunología , Microbiología , Lipoproteínas , Genética , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes de Fusión , Genética , Alergia e Inmunología , Vacunas Sintéticas , Alergia e Inmunología
14.
Chinese Journal of Hepatology ; (12): 522-525, 2003.
Artículo en Chino | WPRIM | ID: wpr-305864

RESUMEN

<p><b>OBJECTIVE</b>To research the value of hepatic perfusion with multi-slice spiral CT in the diagnosis of liver diseases.</p><p><b>METHODS</b>Among the 48 patients undergone dynamic CT of the liver, 20 were volunteers without hepatic disorder, 17 with cirrhosis, 11 suffered from hepatic cancer. The perfusion indexes were calculated and compared.</p><p><b>RESULTS</b>(1) Compared with the control group, HPP (ml/min/ml), PPI and HPP/HAP of patients with cirrhosis were significant lower (HPP: 0.49+/-0.19 vs 0.60+/-0.16, P=0.038; PPI: 0.58+/-0.14 vs 0.67+/-0.06, P=0.015; HPP/HAP: 1.63+/-0.87 vs 2.12+/-0.65, P=0.04), whereas HPI was higher (0.42+/-0.14 vs 0.33+/-0.06, P= 0.015), which indicated the decrease of portal inflow and the increase of arterial inflow in cirrhosis patients. (2) Patients with hepatic cancer got a significant higher average HAP than that in volunteers and cirrhosis patients (F=11.71, P<0.001), while their HPP and HPP/HAP showed significant declining (F=22.84, P=0.0001; F=20.67, P<0.0001, respectively), which implied that hepatic cancer was mainly supplied by artery.</p><p><b>CONCLUSIONS</b>Hepatic perfusion with multi-slice spiral CT is an non-invasive technique to evaluate the arterial and portal inflow separately, which can inflect the hemodynamic change of the lesion by the perfusion indexes, and identify the condition of the tissue round the lesion prior to morphologic change. This method shows important value of diagnosis and differential diagnosis in hepatic diseases.</p>


Asunto(s)
Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Carcinoma Hepatocelular , Diagnóstico por Imagen , Circulación Hepática , Cirrosis Hepática , Diagnóstico por Imagen , Neoplasias Hepáticas , Diagnóstico por Imagen , Proyectos Piloto , Tomografía Computarizada Espiral , Métodos
15.
Chinese Journal of Medical Genetics ; (6): 542-543, 2003.
Artículo en Chino | WPRIM | ID: wpr-329415

RESUMEN

<p><b>OBJECTIVE</b>To investigate the possible association between polymorphism of CC16 gene exon 1 and asthma, the genotype and allele frequencies of CC16 gene exon 1 in the asthmatic patients of Han population in southwest China were analyzed.</p><p><b>METHODS</b>The authors determined the genotypes of CC16 gene exon 1 with polymerase chain reaction technique and restricted enzyme analysis, and then compared the genotype and allele frequencies of the gene of the asthmatic group with those of the healthy control group.</p><p><b>RESULTS</b>There was no significant difference in genotype and allele frequencies of CC16 gene between the asthmatic group and control group. There was no association between the genotype and allele frequencies of gene and the severity of asthma.</p><p><b>CONCLUSION</b>CC16 gene may be not a susceptibility gene of asthmatic patients of Han population in southwest China.</p>


Asunto(s)
Adulto , Anciano , Humanos , Persona de Mediana Edad , Alelos , Asma , Genética , China , Etnología , Predisposición Genética a la Enfermedad , Genotipo , Mutación , Proteínas , Genética , Uteroglobina
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