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BACKGROUND:Generaly, the stent surface modification, especialy seeding cels, may accelerate or cause stent endothelium, and cause restenosis for prevention of in-stent thrombosis. OBJECTIVE: To develop the optimal conditions for vascular stents coated with bone marrow mesenchymal stem cels. METHODS: Cerebrovascular stent was co-cultured with passage 3 bone marrow mesenchymal stem cels from rats at 1×106, 1×107, 1×108, and 1×109/L. Cels on the stents were examined with transmission electron microscopy after 48 hours. A total of 160 male Sprague-Dawley rats were enroled, among which, 20 rats were as normal control group, and the remaining 140 were used for producing models of ischemic stroke that were randomly sub-divided into seven groups at 8 weeks after modeling: stainless steel stent implanted group, polymer stent group, and different concentrations of cellstent composite groups. After 8 weeks of implantation, the expression of vascular endothelial growth factor in these cels was examined by western blot assay. Rat platelet activation in different groups was determined by flow cytometry. RESULTS AND CONCLUSION:Implanted stem cels were able to grow adherently on the stainless steel stent wal. When the planting cellconcentration was 1×107 cels/L, the cels and organeles were morphologicaly normal and covered the stent surface wel. These coated cels also expressed vascular endothelial growth factor, suggesting that they functioned as endothelial cels, and they also significantly lowered platelet activation. When co-cultured with 1×107/L bone marrow mesenchymal stem cels, the stent was covered wel with endothelial-like cels and had significant lower platelet activationin vivo.
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The thrombolytic agent DSPAalpha1 is currently undergoing clinical trials for the treatment of acute ischemic stroke and has shown good pharmacodynamic, pharmacokinetic and safety profiles. Here, the DSPAalpha1 gene, optimized for the preferred codons of yeast, was cloned into the Pichia pastoris strains GS115 and KM71. Both expression systems produced functional DSPAalpha1 into the broth medium under shaking flask growth conditions with the yield of about 70 mg/L, and 105 mg/L, respectively. In addition, three glycosylation minus DSPAalpha1 mutants, constructed by site-directed mutagenesis, were also expressed in Pichia pastoris. The mutant proteins were assayed by SDS-PAGE and fibrin degradation activities were evaluated. The secretion levels of all the mutants, especially N362Q and N117Q/N362Q, were so lower compared to the wild-type DSPAalpha1 that only minimal quantities of mutant protein could be recovered by purification from the culture medium. The protein specific activities from mutants (N117Q, N362Q) were less 25% than that of the wild type protein. These results imply that the N-linked carbohydrate chains (at N117 and N362) are vital for the enzymatic activity of rDSPAalpha1 and for its secretion from Pichia pastoris.
Subject(s)
Fibrinolytic Agents , Metabolism , Genetic Vectors , Genetics , Glycosylation , Pichia , Genetics , Metabolism , Plasminogen Activators , Genetics , Recombinant Proteins , Genetics , PharmacologyABSTRACT
AIM: To quantify magnesium isoglycyrrhizinate(MGL) and glycyrrhetic acid in the plasma of dog by develop a simple, rapid, sensitive high-performance liquid chromatography (HPLC-UV) method. METHODS: HPLC-UV methods with wavelength 252 nm were used for the quantitation of MGL and GA in plasma. The concentration of MGL and GA were assayed on a Kromasil ODS-1 C18 column with the column temperature 25 ℃. The mobile phase was a gradient system with 0.1 % diethylamine in water (pH 4.60 ) and acetonitrile at a flow rate of 1.0 ml?min~ -1 . RESULTS: There was a good linear response range of 0.2 -2.5 mg?L~ -1 and 2.5 -100 mg?L~ -1 , while the limit of quantification was 0.2 mg?ml~ -1 . The relative recovery rate of MGL was 94.3 %-101.9 %,GA 96.4 %-101.9 % (n=5);the absolute recovery rate was MGL 78.7 %-87.0 %, GA 77.5 %-87.7 % (n=5). The intra-day and inter-day variations were all less than 15% (n=5). The plasma samples preserved in refrigerator is stable at -20 ℃ for 14 days, freeze thawing 3 times and at room temperature for 10 h without degradation. CONCLUSION: This method is shown to be specific, sensitive, reliable and suitable for the quantitative determination of magnesium isoglycyrrhizinate following oral administration in biological sample.
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ObjectiveTo construct the eukaryotic coexpression plasmid CEA/IL-2, and to lay the foundation for further studying CEA nucleiotide vaccine , adjuvant and their effects of special antitumor immunity.MethodsThe eukaryotic expression plasmid (pcDNA3-CEA)containing the gene coding for CEA was obtained by RT-PCR and gene recombination techniques.Using enzymolysis,ligation and other techniques,an eukaryotic coexpression plasmid (pIRES-CEA/IL-2)containing two expression unites of CEA and IL-2 gene connected with internal ribosome site was constructed.ResultsThe coexpression plasmids were transformed into COS7 cells and expression of two proteins were demonstrated by ELISA, and flow cytometer and elecsy.CEA and IL-2 were (23.73?0.26)ng/ml,and(20.17?0.13)ng/ml respectively.ConclusionsThe eukaryotic expression plasmids pIRES-CEA/IL-2 could be successfully constructed and transformed into COS7 cells.Expression of two proteins were demonstrated with no difference on expression.
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Objective: To clone porcine IL-4 cDNA and observe its adjuvanticity of vaccine against T. solium cysticerco-sis. Methods:The cDNA of porcine IL-4 was amplified by RT-PCR, which had 5' AUG initiatory codon with optimized trans-lational initiation. After sequencing,the cDNA was intergrated into expression vector pcDNA and transient expression was performed. Then newborn pigs were immunized with IL-4 expression vector and protective antigen DNA vaccine against T. solium cysticercosis. Results:The obtained sequence of porcine IL-4 cDNA was the same as reported. IL-4 and protective antigen could effectively promote the humoral response. Conclusion:An efficient expression plasmid containing porcine IL-4 cDNA is constructed. Its adjuvanticity of DNA vaccine against T. solium cysticercosis is preliminarily conformed.
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Objective To clone and express the gene encoding ESAT antigen of Mycobacterium tuberculosis (chinese stain). Methods Genome of mycobacterium tuberculosis was extracted. Then, the full length cDNA encoding ESAT protein was amplified by PCR and cloned into prokaryotic expressing vector pGEX5T and sequenced. pGEX5T ESAT was expressed in k802 Ecoli, and the expressed protein was determined by western blot using the sera from ten patients with tuberculosis. Results One specific band of 0.3kb or so was obtained and sequencing result was identical to that reported from Genbank. The expressed protein could be specifically recognized by the sera from tuberculosis patients. Conclusions The full length cDNA of Mycobacterium tuberculosis (Chinese strain) ESAT protein was cloned and expressed successfully, which will be helpful in further studies on diagnosis and treatment of tuberculosis.
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Objective To analyse the immunostimulatory activity of CpG sequences in cysticercus cellulosae paramyosin (also named Antigen B,AgB)cDNA. Methods C57BL/6 mice were immunized with pcDNA3 AgB plasmid,pcDNA3 AgB′(CpG sequences were mutated),pcDNA3 or AgB protein and two weeks later,immune response was assayed by ELISA. Results IgG and IgG 2a were detectable at week 2 after immunization and continually increased until week 4.The antibody levels elicited by pcDNA3 AgB were significantly higher( P
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Objective: To fuse Mycobacterium tuberculosis protective antigen gene with mice ubiquitin gene, constructing antigen ubiquitin system. Methods: Mice ubiquitin cDNA was amplified by RT PCR from mice testicle,and 4 antigen genes were obtained by PCR from cultured Mycobacterium tuberculosis . Ubiquitin and 4 antigen genes were linked by flexible adaptor respectively and the fusing genes were cloned into pVAX vector.The recombinant plasmids were digested with endonuclease and sequenced.Then the recombinant plasmids were transfected into COS7 cells and the expression was assayed by ELISA. Results:Ubiquitin and 4 antigen genes were 0.2,0.3,0.7,1.0,1.65 kb in length by agarose electrophoresis. Endonuclease digestion of the recombinant plasmids indicated that the fusion genes were correctly inserted into pVAX vector. Sequencing results of fusion nucleic acid vaccines were identical to those in GenBank.The recombinant plasmids expressed in COS7 cells. Conclusion: Four Chinese Mycobacterium tuberculosis protective antigens ubiquitin systems are successfully constructed and can be expressed in eukaryotic cells. This may provide a basis for diagnosis and therapy of tuberculosis.