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Objective:To explore the effect of epalrestat combined with calcium dobesilate on cardiovascular and cerebrovascular diseases and oxidative stress in diabetes patients with peripheral neuropathy.Methods:In a retrospective analysis, 92 patients with diabetic peripheral neuropathy were admitted to the Department of Endocrinology and Metabolism Hospital of Bozhou People’s Hospital from Jun. 2018 to Jun. 2021, which were divided into observation group and control group according to the different treatment methods, with 46 cases in each group. The control group were treated with epalrestat. On this basis, the observation group were given calcium dobesilate combined treatment. The two groups were compared in terms of the Michigan diabetes neuropathy score (MDNS) Michigan neuropathy screening instrument (MNSI) score, oxidative stress reaction [serum superoxide dismutase (SOD), reduced glutathione (GSH) ], clinical efficacy, complications and adverse reactions Using t-test and chi square test.Result:After treatment, the MDNS and MNSI scores of both groups decreased, and the observation group [ (23.49±3.73), (8.49±1.97) ] were lower than the control group [ (28.49±3.85), (11.53±2.28) ] ( t=8.337, 6.843, P<0.05) ; After treatment, the levels of SOD and GSH in both groups increased compared to those before treatment, and the observation group [ (38.96±4.89), (89.79±6.92) ] were higher than the control group those [ (36.42±4.61), (86.74±6.20) ] ( t=2.563, 2.226, P=0.012, 0.028) ; The clinical efficacy of the observation group was higher than that of the control group ( Z=1.592, P=0.042) ; The incidence of complications in the observation group after 1 year was significantly lower than that in the control group ( χ2=4.389, P=0.036) ; The incidence of adverse reactions in the observation group was slightly lower than that in the control group ( χ2=0.155, P=0.694) . Conclusion:Epalrestat combined with calcium dobesilate is effective in the treatment of diabetes peripheral neuropathy, can effectively reduce complications, and has high treatment safety, which is worthy of clinical application.
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Toll-like receptor 4 (TLR4), a type Ⅰ transmembrane protein, has been extensively studied in the Toll-like receptor family at present. TLR4 ligands include lipopolysaccharides ( LPS) present in the outer membrane of gram-negative bacteria and monophosphoryl lipid A ( MPLA) which is a derivative of LPS. TLR4 agonists, alone, as a major component of compound adjuvants or in combination with other TLRs agonists, have been widely used as adjuvants in various vaccines and demonstrated great potential in vaccine development. This review addressed the discovery, application, features and prospect of novel vac-cine adjuvants based on TLR4 agonists, aiming to provide reference for rational use of adjuvants and further development.
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Objective To compare the adjuvant activity of oil-in-water emulsion MF59 and aluminum hydroxide on immune responses to HIV-1 multi-epitope protein MEP1 in BALB/c mice. Methods HIV-1 multi-epitope protein MEP1 with MF59 or aluminum was prepared to intramuscularly vaccinate female BALB/c mice for three times. Serum and splenocytes were isolated from the mice 10 d after the first and last vaccination. Specific anti-MEP1 antibodies and the subclasses in serum were detected by ELISA. The num-bers of splenic lymphocytes secreting IFN-γ and IL-4 were measured by ELISPOT. Differences in humoral and cellular immune responses induced by the two different adjuvants were compared. Results After a sin-gle-dose immunization, aluminum adjuvant promoted early humoral and cellular immune responses to MEP1 compared with MF59. After the three-dose immunization, both aluminum adjuvant and MF59 promoted MEP1 to induce Th2-biased humoral immune response, while MF59 also enhanced a balanced Th1/Th2 cel-lular immune response. Conclusions Aluminum adjuvant was a more suitable adjuvant than MF59 for HIV-1 multi-epitope protein MEP1.
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Objective To express a recombinant protein TFPR1 ( the functional region of the snake venom proteins from Trimeresurus flavoviridis) in Pichia pastoris expression system. Methods The target gene was codon-optimized and synthesized according to the sequence of the conserved structural do-main of triflin and then cloned into the Pichia pastoris expression vector pPICZαA to construct the recombi-nant expression plasmid pPICZαA-TFPR1. The recombinant plasmid pPICZαA-TFPR1 was electroporated into the yeast strain X33. The transformed strains carrying expression plasmid were screened out with Zeocin and then induced by methanol to express the recombinant protein TFPR1. ELISA was performed for the screening of positive clones. SDS-PAGE and Western blot were used for further identification of the ex-pressed products. Results The recombinant plasmid pPICZαA-TFPR1 was successfully constructed. The recombinant protein TFPR1 was expressed in a secreted form at a molecular weight of 16×103. Conclusion The recombinant protein TFPR1 was successfully expressed in Pichia pastoris expression system, which laid a foundation for further researches on its biological function and application as an adjuvant.
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Objective To establish a simple and quick loop-mediated isothermal amplification (LAMP)method for detection of Yersinia pestis.Methods LAMP Primers were designed based on the specific sequence 3a in Y.pestis chromosome.LAMP reaction results were detected using turbidity meter or visual method.The specificity of the constructed method was evaluated by detecting Y.pestis and its closely-related bacteria.The different dilution DNA template was detected with LAMP and PCR to evaluate the sensitivity of the method.Results Thirty strains of bacteria closely related to Y.pestis were detected by the constructed LAMP,and all the results were negative,indicating that the method had a very high specificity.The detection sensitivity of this LAMP assay was 20 pg of DNA per reaction,which was ten-fold that of the regular PCR.The detection reaction was completed in 25 min.Conclusion This LAMP method is quick,sensitive, specific and simple,which is expecked to become an effective method for rapid detection of Y.pestis on the scene.
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Quantitative specific detection of Staphylococcus aureus is based on recombinant lysostaphin and ATP bioluminescence. To produce recombinant lysostaphin, the lysostaphin gene was chemically synthesized and inserted it into prokaryotic expression vector pQE30, and the resulting expression plasmid pQE30-Lys was transformed into E. coli M15 for expressing lysostaphin with IPTG induction. The recombinant protein was purified by Ni(2+)-NTA affinity chromatography. Staphylococcus aureus was detected by the recombinant lysostaphin with ATP bioluminescence, and plate count method. The results of the two methods were compared. The recombinant lysostaphin was successfully expressed, and a method of quantitative specific detection of S. aureus has been established, which showed a significant linear correlation with the colony counting. The detection method developed has good perspective to quantify S. aureus.
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Trifosfato de Adenosina , Química , Cromatografia de Afinidade , Escherichia coli , Medições Luminescentes , Métodos , Lisostafina , Química , Proteínas Recombinantes , Química , Staphylococcus aureusRESUMO
OBJECTlVE To study the anti-HBV activity of prepared recombinant human serum aIbu-min-interferon α-2b fusion protein(HSA-IFNα-2b) in vitro. METHODS HepG2 ceIIs were infected with recombinant adenovirus with green fIuorescence protein and 1.6-foId HBV DNA(AdGFP-HBV). The ex-pression of HBV antigens,HBsAg and HBeAg in cuIture medium was detected by ELISA assay. The tox-icity of HSA-IFNα-2b on HepG2 ceIIs was evaIuated by mTT assay.The reIative expression of HBV RNA in ceIIs and the absoIute quantity of HBV DNA in cuIture supernatant were determined by quantitative PCR assay. The activity of HBV enhancer Ⅰ was detected by DuaI-Reporter gene assay. RESULTS HBV couId repIicate and express in HepG2 ceIIs after infection with AdGFP-HBV. The expression of HBsAg and HBeAg in cuIture serum of HepG2 ceIIs infected with AdGFP-HBV decreased by 51.32%(P﹤0.01)and 50.26%(P﹤0.01),respectiveIy,when HSA-IFNα-2b 500 kU·L-1 was added. The same concentration of HSA-IFNα-2b didn't inhibit the proIiferation of HepG2 ceIIs,but inhibited HBsAg in a concentration-dependent manner. The regression formuIa between HBsAg inhibitory rate(Y)and con-centration of HSA-IFNα-2b(X)was Y=21.11 IgX+11.91(r 2 = 0.954),IC50 = 63.76 kU·L-1 . HBV RNA in ceIIs and HBV DNA in the cuIture serum decreased by 52.83%(P﹤0.01)and 53.07%(P﹤0.01), respectiveIy,when HSA-IFNα-2b 500 kU·L-1 was added. The activity of enhancer Ⅰ decreased by 40.04%(P﹤0.01)when HSA-IFNα-2b 500 kU·L-1 was added. CONCLUSlON The ceII modeI of HBV repIication for evaIuating anti-HBV agents is successfuIIy estabIished. HSA-IFNα-2b exhibits noticeabIe anti-HBV effect invitro.
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Objective To establish a mouse model of acute liver failure induced by lipopolysaccharide /D-galac-tosamine ( LPS/D-GalN) .Methods The optimum dose of LPS/D-GalN was determined by i .p.injection of eight differ-ent doses of LPS and D-GalN into 40 female C57BL/6 mice and observation of their survival time .Then, 32 female C57BL/6 mice were i.p.injected with the optimal dose of LPS/D-GalN and sacrificed at 0, 1, 4, 8 hours after the injec-tion, 8 mice in each group.The control mice received saline injection .Hepatic changes were observed by pathology and se-rum ALT, IL-6, MCP-1 and TNF-αwere measured by biochemistry or flow cytometry .Results LPS (2.5 mg/kg) and D-GalN (0.3 g/kg) were determined as the optimal dose for the establishment of mouse model of acute liver injury .Com-pared with the control group , the hepatocellular damages were progressing in a positive correlation with the time course after LPS/D-GalN administration .The level of serum ALT was significantly increased after LPS/D-GalN administration ( P <0.001).The levels of inflammatory cytokines IL-6, MCP-1 and TNF-αwere increased and reached a peak at one hour after LPS/D-GalN administration and then decreased almost to that of the control group 8 hours later(P<0.001).Conclusions The mouse model of acute liver injury is successfully established by LPS /D-GalN administration , and provide an effective animal model for the study of pathogenic mechanisms of acute liver failure and evaluation of therapeutic drugs .
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Objective To analyze and compare the pathological changes of lung tissue in mice infected with the novel H7N9 influenza virus and 2009 pandemic H1N1 influenza virus, respectively, and to preliminarily study the mecha-nisms of acute lung injury induced by those virus infection .Methods SPF 6-week old BALB/c mice ( body weight 18-20 g, male∶female=1∶1) (n=3 in each subgroup) were intranasally infected with H7N9 virus and H1N1 virus, respec-tively.The behavior and survival time of mice after virus infection were observed and the survival rates were analyzed .The heart, liver, spleen, lung, kidney, intestines, and brain were collected at indicated time points for histopathological exami-nation using H&E staining .The distribution of virus antigen was detected by immunohistochemistry .The neutrophil infiltra-tion was also observed .The correlation of lung injury with virus replication and host immune responses was analyzed .Re-sults The lung and spleen injury of mice infected with H 7N9 virus was slighter and their survival rate (100%) was high-er than those of mice infected with H1N1 virus.The damages of the lung and spleen in H1N1virus-infected mice were more severe than that in H7N9 virus-infected mice, and all the 10 mice in this group died within 9 days after virus inoculation . The distributions of both the virus antigens were mainly in the bronchial epithelial cells , a few stromal cells and alveolar ep-ithelial cells .The levels of virus replication in the two groups were not significantly different .There were more intense neu-trophil infiltration in the lung and inflammatory response in the H 1N1 virus-infected mice than those in the H7N9 virus-in-fected mice .Conclusions There are some differences of the pathological characteristics and extent of lung injury in the mice infected with H7N9 virus and H1N1 virus, respectively.The virus replication is a precipitating factor but not the deci-sive factor of the lung injury , and there is a close relationship between the host immune responses and acute lung injury .
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<p><b>OBJECTIVE</b>To construct a dual-reporter gene system that will be applicable for use as a tool to screen and evaluate therapeutic drug compounds that inhibit transcription of the gene encoding collagen I, chain at1 (COL1A1).</p><p><b>METHODS</b>The full-length eDNA of transforming growth factor beta1 (TGFbeta1) was cloned by RT-PCR and inserted into two vectors, pcDNA3.1 and pJW4303, for construction of two eukaryotic expression vectors, pcDNA3.1-TGFbeta1 and pJW4303-TGFbeta1.Next, the promoter region of COL1A1, cloned by PCR using human genome DNA as template, was inserted into the vector pGL4.29 to construct the reporter gene vector, pGL4.29-COL1A1 promoter.All three recombinant vectors were verified by restriction enzyme digestion and DNA sequencing.Either the pcDNA3.1-TGFbeta1 or pJW4303-TGFbeta1 vector along with the pGL4.29-COL1A1 promoter vector or a pRL-null, control reporter, vector were co-transfected into the LX-2 human hepatic stellate cells to establish the transcription-activated dualreporter gene system.This system was used as a cell model for screening anti-liver fibrosis compounds that inhibit the transcription of COL1A1.Dexamethasone, a model drug that is known to inhibit the expression of COL1A1, was used as a control to validate the dual-reporter gene system.</p><p><b>RESULTS</b>The two TGFbeta1-expressing vectors and the reporter gene vector containing the promoter region of COL1A1 were successfully constructed.The results of a dual-reporter gene assay showed that TGFbeta1 co-expression increased the activity of the COL1A1 promoter by above 200-fold (t =21.78, P =0.0001), whereas in the absence of TGF31 co-expression the activity was below 2-fold (t =3.396, P =0.0274).The transcriptionactivated dual-reporter gene system was successfully established.The model drug, dexamethasone, effectively inhibited the activity of the COL 1A1 promoter in dose-dependent manner; the activity decreased 29.6% with 10 mumol/L dexamethasone (t =4.140, P =0.0144) and 53.9% with 100 mumol/L (t =6.193, P =0.0035).</p><p><b>CONCLUSION</b>The dual-luciferase reporter system of TGFbeta1 and COL1A1 co-expression developed here can be used as a cell model to screen and evaluate anti-liver fibrosis compounds that inhibit activity of the COL1A1.</p>
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Humanos , Sequência de Bases , Colágeno Tipo I , Genética , Avaliação Pré-Clínica de Medicamentos , Genes Reporter , Vetores Genéticos , Cirrose Hepática , Tratamento Farmacológico , Luciferases , Regiões Promotoras Genéticas , Ativação Transcricional , Transfecção , Fator de Crescimento Transformador beta1RESUMO
A simple rapid detection of antibody to hepatitis delta virus (anti-HDV) in human serum was developed by using double antigen sandwich ELISA. HDV gene fragment encoding HDAg was isolated from a Chinese patient infected with HDV by RT-PCR, and a high-efficient expression HD-PQE31 strain was constructed with the fragment. We obtained high titer and good quality hepatitis delta virus protein purified by Ni-NTA metal-affinity chromatography, which was identified by Western blot and ELISA, then we set up the double antigen sandwich ELISA for detection of anti-HDV in human serum, and the performance of the sandwich ELISA was evaluated in terms of specificity and sensitivity. Results were: 1) The purified HDAg protein's purity was 90%, and its ELISA titer was 1/100 000. 2) 42 anti-HDV positive sera were detected and showed that the sensitivity of sandwich ELISA was higher than that of competitive ELISA (t=2.44, p<0.01). 3) The inhibitory rates for 2 anti-HDV positive sera by the specific HDAg were 74% and 93% respectively. 4) For the assay of specificity, all 60 samples infected by other hepatitis viruses and 30 normal samples were negative for anti-HDV. These results suggested that the double antigen sandwich ELISA with purified recombinant HDAg showed higher specificity and sensitivity, It can be used in routine laboratories to diagnose the HDV infection.
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Objective To investigate the effects of glutamate receptor signaling on melanoma cell dendrite morphology and cytoskeleton protein. Methods A metastatic human malignant melanoma cell line WM451LU was cultured and transfected by recombinant adenovirus vector carrying a cDNA encoding microtubule-associated protein 2a (MAP2a). MK-801, an antagonist of N-methyl-D-aspartate receptor (NMDAR), and CPCCOEt, an antagonist of metabotropie glutamate receptor 1 (mGluR1), were used to treat transfected or untrausfeeted WM451LU cells. Confocal microscopy and three dimensional atomic force microscopy were used to assess subcellular location of NMDAR2A, mGluR1 and MAP2a as well as the dis-tribution of α-tubulin in and dendrite morphology of WM451LU cells. The proliferation of WM451LU cells was estimated by cell survival growth curve. Results Confocal laser microscopy revealed that NMDAR2A, mGluRl and MAP2a were mainly co-localized in melanoma cell dendrites. Both MK-801 and CPCCOEt increased the density of microtubules in cell dendrites and dendritic branching of WM451LU cells, and both effects of MK-801 and CPCCOEt were enhanced by the expression of MAP2a. Furthermore, the proliferation of WM451LU cells was significantly inhibited by MK-801 of 100 μmol/L and CPCCOEt of 10 μmol/L. Conclusions In melanoma cells, glutamate receptors may participate in the development of dendrites, and anta- gonists of glutamate receptors could inhibit the proliferation of melanoma cells.
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Abstract: To express human-mouse chimeric IgA antibody directed against H5N1 virus, an anti-H5N1 chimeric IgA antibody gene was constructed by joining the light and heavy chain variable region genes and the corresponding signal peptide coding sequences of the anti-H5N1 mouse monoclonal antibody H5N1-HA with the coding sequences of the constant region of the human IgA2 heavy chain and Kappa chain respectively. Then the full-length chimeric light and heavy chain expressing plasmids pEF-IGHA9 and pEF-IGK9 were constructed and transfected into the CHO/dhfr cells. The chimeric IgA antibody expression was confirmed by ELISA, SDS-PAGE and Western blotting. The successful expression of this anti-H5N1 chimeric IgA may help to provide a stand for developing passive immunological agents for H5N1 virus infection prophylaxis.
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Animais , Cricetinae , Humanos , Camundongos , Anticorpos Monoclonais , Anticorpos Antivirais , Genética , Células CHO , Quimerismo , Cricetulus , Imunoglobulina A , Genética , Alergia e Imunologia , Virus da Influenza A Subtipo H5N1 , Genética , Alergia e Imunologia , Proteínas Recombinantes de Fusão , Genética , Alergia e ImunologiaRESUMO
To use the designed restriction enzyme assisted mutagenesis technique to perform rapid site-directed mutagenesis on double-stranded plasmid DNA. The target amino acid sequence was reversely translated into DNA sequences with degenerate codons, resulting in large amount of silently mutated sequences containing various restriction endonucleases (REs). Certain mutated sequence with an appropriate RE was selected as the target DNA sequence for designing mutation primers. The full-length plasmid DNA was amplified with high-fidelity Phusion DNA polymerase and the amplified product was 5' phosphorylated by T4 polynucleotide kinase and then self-ligated. After transformation into an E. coli host the transformants were rapidly screened by cutting with the designed RE. With this strategy we successfully performed the site-directed mutagenesis on an 8 kb plasmid pcDNA3.1-pIgR and recovered the wild-type amino acid sequence of human polymeric immunoglobulin receptor (pIgR). A novel site-directed mutagenesis strategy based on DREAM was developed which exploited RE as a rapid screening measure. The highly efficient, high-fidelity Phusion DNA polymerase was applied to ensure the efficient and faithful amplification of the full-length sequence of a plasmid of up to 8 kb. This rapid mutagenesis strategy avoids using any commercial site-directed mutagenesis kits, special host strains or isotopes.
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Sequência de Aminoácidos , Sequência de Bases , DNA , Genética , Enzimas de Restrição do DNA , Genética , DNA Polimerase Dirigida por DNA , Genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Métodos , Plasmídeos , Receptores de Imunoglobulina Polimérica , GenéticaRESUMO
Objective To study the immunoreaction of the recombinant proteins encoded by the fragments of ORF2 ( second open reading frames) gene of hepatitis E virus ( HEV). Methods The aimed sequence from the full-length ORF2 in clone PEH2 , which was derived from a Chinese strain of HEV,was amplified and cloned it into vector pcDNAS. 1 which was then transfected to 293 cell. Results The ORF2 protein was present in the soluble fractions of the cell lysate. The expressed protein of HEV ORF2 in 293 cells by using a plasmid pcDNA3. 1-based system showed positive on immunoblots probed against antibodies raised in BALB/c mice. Conclusion The experimental results laid a foundation for developing diagnostic reagent to detect HEV by using the expressed products of ORF2 protein.
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Objective To obtain Chinese hamster ovary (CHO) cell lines that stably express a targeting complement inhibitor CR2-CD59.Methods The recombinant plasmid PEE14.1-CR2-CD59 was constru-cted by cloning the DNA fragment CR2-CD59 into plasmid PEE14.1,and the obtained plasmid was transfected into CHO cells by FuGENE 6.The clones with stable high expression of target fragment were selected by methionine sulfoximine (MSX),the expression of CR2-CD59 was analyzed by ELISA,SDS-PAGE and Western blotting analysis.Results Several stable expression clones were obtained,and CR2-CD59 was highly expressed in the secret form in CHO cells.SDS-PAGE analysis showed that the molecular weight of the recombined protein CR2-CD59 was consistent with the predicted one.ELISA and Western blotting results revealed that the CR2-CD59 could react with both anti-human CR2 and anti-human CD59 polyclonal antibodies.Compared with serum-containing medium,the protein was highly expressed in serum-free medium (P
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<p><b>OBJECTIVE</b>To study the pathological characteristics of severe acute respiratory syndrome (SARS) and its relationship to clinical manifestation.</p><p><b>METHODS</b>Tissue specimens from 3 autopsies of probable SARS cases were studied by microscope, and the clinical data was reviewed.</p><p><b>RESULTS</b>The typical pathological changes of lungs were diffuse hemorrhaging on the surface. A combination of serous, fibrinous and hemorrhagic inflammation was seen in most of the pulmonary alveoli with the engorgement of capillaries and detection of micro-thrombosis in some of these capillaries. Pulmonary alveoli thickened with interstitial mononuclear inflammatory infiltrates, suffered diffuse alveolar damage, experienced desquamation of pneumocytes and had hyaline-membrane formation, fibrinoid materials, and erythrocytes in alveolar spaces. There were thromboembolisms in some bronchial arteries. Furthermore, hemorrhagic necrosis was also evident in lymph nodes and spleen with the attenuation of lymphocytes. Other atypical pathological changes, such as hydropic degeneration, fatty degeneration, interstitial cell proliferation and lesions having existed before hospitalization were observed in the liver, heart, kidney and pancreas.</p><p><b>CONCLUSION</b>Severe damage to the pulmonary and immunological systems is responsible for the clinical features of SARS and may lead to the death of patients.</p>