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The human tumor necrosis factor α (hTNF-α) is an important pro-inflammatory cytokine which plays critical roles in inflammatory diseases such as rheumatoid arthritis (RA). The anti-TNF-α proteins can reduce symptoms of RA. Due to limitations of protein-based therapies, it is necessary to find new anti-TNF-α agents instead of common anti-TNF-α proteins. Therefore, the aim of the current study was to identify a new DNA aptamer with anti-hTNF-α activity. The protein systematic evolution of ligands by exponential enrichment (SELEX) process was used for identifying DNA aptamers. Anti-hTNF-α aptamers were selected using dot blot, real-time PCR, and in vitro inhibitory assay. The selected aptamers were truncated in two steps, and finally, a dimer aptamer was constructed from different selected truncates to improve their inhibitory effect. Also, Etanercept was used as a positive control to inhibit TNF-α, in comparison to the designed aptamers. After 11 rounds, four aptamers with anti-hTNF-α inhibitory effect were identified. The truncation and dimerization strategy revealed a new dimer aptamer with 67 nM Kd, which has 40% inhibitory effect compared with Etanercept (60%). Overall, the dimerization and truncation aptamers could improve its activity. With regard to the several limitations of anti-TNF-α proteins therapies including immunogenicity, side effects, and cost-intensive, a new designed anti-hTNF-α dimer aptamer could be considered as a potential therapeutic and/or diagnostic agent for hTNF-α-related disorders. 相似文献
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The misfolding of specific proteins is often associated with their assembly into fibrillar aggregates, commonly termed amyloid fibrils. Despite the many efforts expended to characterize amyloid formation in vitro, there is no deep knowledge about the environment (in which aggregation occurs) as well as mechanism of this type of protein aggregation. Alpha-chymotrypsin was recently driven toward amyloid aggregation by the addition of intermediate concentrations of trifluoroethanol. In the present study, approaches such as turbidimetric, thermodynamic, intrinsic fluorescence and quenching studies as well as chemical modification have been successfully used to elucidate the underlying role of hydrophobic interactions (involved in early stages of amyloid formation) in α-chymotrypsin-based experimental system. 相似文献
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Russian Journal of Plant Physiology - Buxus sempervirens L. is an endangered ornamental shrub in the Hyrcanian forests in the northern part of Iran. B. sempervirens has excellent ornamental value.... 相似文献
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Kazem Mashayekhi Mojtaba Sankian Saeed Mohammadian Haftcheshmeh Ramezan Ali Taheri Kazem Hassanpour Gholamreza Farnoosh 《Biotechnology progress》2021,37(5):e3191
To increase the efficiency of aptamers to their targets, a simple and novel method has been developed based on aptamer oligomerization. To this purpose, previously anti-human TNF-α aptamer named T1–T4 was trimerized through a trimethyl aconitate core for neutralization of in vitro and in vivo of TNF-α. At first, 54 mer T1–T4 aptamers with 5′-NH2 groups were covalently coupled to three ester residues in the trimethyl aconitate. In vitro activity of novel anti-TNF-α aptamer and its dissociation constant (Kd) was done using the L929 cell cytotoxicity assay. In vivo anti-TNF-α activity of new oligomerized aptamer was assessed in a mouse model of cutaneous Shwartzman. Anchoring of three T1–T4 aptamers to trimethyl aconitate substituent results in formation of the 162 mer fragment, which was well revealed by gel electrophoresis. In vitro study indicated that the trimerization of T1–T4 aptamer significantly improved its anti-TNF-α activity compared to non-modified aptamers (P < 0.0001) from 40% to 60%. The determination of Kd showed that trimerization could effectively enhance Kd of aptamer from 67 nM to 36 nM. In vivo study showed that trimer aptamer markedly reduced mean scar size from 15.2 ± 1.2 mm to 1.6 ± 0.1 mm (P < 0.0001), which prevent the formation of skin lesions. In vitro and in vivo studies indicate that trimerization of anti-TNF-α aptamer with a novel approach could improve the anti-TNF-α activity and therapeutic efficacy. According to our findings, a new anti-TNF-α aptamer described here could be considered an appropriate therapeutic agent in treating several inflammatory diseases. 相似文献
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Efficient generation of smooth muscle cells from adipose‐derived stromal cells by 3D mechanical stimulation can substitute the use of growth factors in vascular tissue engineering
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Mojtaba Parvizi Lydia A.M. Bolhuis‐Versteeg André A. Poot Martin C. Harmsen 《Biotechnology journal》2016,11(7):932-944
Occluding artery disease causes a high demand for bioartificial replacement vessels. We investigated the combined use of biodegradable and creep‐free poly (1,3‐trimethylene carbonate) (PTMC) with smooth muscle cells (SMC) derived by biochemical or mechanical stimulation of adipose tissue‐derived stromal cells (ASC) to engineer bioartificial arteries. Biochemical induction of cultured ASC to SMC was done with TGF‐β1 for 7d. Phenotype and function were assessed by qRT‐PCR, immunodetection and collagen contraction assays. The influence of mechanical stimulation on non‐differentiated and pre‐differentiated ASC, loaded in porous tubular PTMC scaffolds, was assessed after culturing under pulsatile flow for 14d. Assays included qRT‐PCR, production of extracellular matrix and scanning electron microscopy. ASC adhesion and TGF‐β1‐driven differentiation to contractile SMC on PTMC did not differ from tissue culture polystyrene controls. Mesenchymal and SMC markers were increased compared to controls. Interestingly, pre‐differentiated ASC had only marginal higher contractility than controls. Moreover, in 3D PTMC scaffolds, mechanical stimulation yielded well‐aligned ASC‐derived SMC which deposited ECM. Under the same conditions, pre‐differentiated ASC‐derived SMC maintained their SMC phenotype. Our results show that mechanical stimulation can replace TGF‐β1 pre‐stimulation to generate SMC from ASC and that pre‐differentiated ASC keep their SMC phenotype with increased expression of SMC markers. 相似文献