RESUMEN
Cornea is an avascular and transparent tissue that focuses light on retina. Cornea is supported by the corneal-endothelial layer through regulation of hydration homeostasis. Restoring vision in patients afflicted with corneal endothelium dysfunction-mediated blindness most often requires corneal transplantation (CT), which faces considerable constrictions due to donor limitations. An emerging alternative to CT is corneal endothelium tissue engineering (CETE), which involves utilizing scaffold-based methods and scaffold-free strategies. The innovative scaffold-free method is cell sheet engineering, which typically generates cell layers surrounded by an intact extracellular matrix, exhibiting tunable release from the stimuli-responsive surface. In some studies, scaffold-based or scaffold-free technologies have been reported to achieve promising outcomes. However, yet some issues exist in translating CETE from bench to clinical practice. In this review, we compare different corneal endothelium regeneration methods and elaborate on the application of multiple cell types (stem cells, corneal endothelial cells, and endothelial precursors), signaling molecules (growth factors, cytokines, chemical compounds, and small RNAs), and natural and synthetic scaffolds for CETE. Furthermore, we discuss the importance of three-dimensional bioprinting strategies and simulation of Descemet's membrane by biomimetic topography. Finally, we dissected the recent advances, applications, and prospects of cell sheet engineering for CETE.
RESUMEN
Vibrio cholerae is the causative agent of cholera and annually leads to death of thousands of people around the globe. Two factors in the pathogenesis of this bacterium are its pili and flagella. The main subunits of pili TcpA, TcpB, and FlaA are the constituent subunit of flagella. In this study, we studied the ability of pili and flagella subunits to stimulate immune responses in mice. After amplification of TcpA, TcpB, and FlaA genes using PCR, they were cloned in expression plasmids. After production of the above-mentioned proteins by using IPTG, the proteins were purified and then approved using immunoblot method. After injection of the purified proteins to a mice model, immune response stimulation was evaluated by measuring the levels of IgG1 and IgG2a antibody titers, IL5 and IFN-γ. Immune response stimulation against pili and flagella antigens was adequate. Given the high levels of IL5 titer and IgG1 antibody, the stimulated immune response was toward Th1. Humoral immune response stimulation is of key importance in prevention of cholera. Our immunological analysis shows the appropriate immune response in mice model after vaccination with recombinant proteins. The high level of IL5 and low level of IFN-γ show the activation of Th2 cell response.
Asunto(s)
Anticuerpos Antibacterianos/sangre , Vacunas contra el Cólera/química , Vacunas contra el Cólera/inmunología , Proteínas Fimbrias/inmunología , Flagelina/inmunología , Animales , Anticuerpos Antibacterianos/inmunología , Cólera/inmunología , Modelos Animales de Enfermedad , Flagelos/inmunología , Humanos , Inmunidad Humoral , Inmunoglobulina G/sangre , Interferón gamma/sangre , Interleucina-5/sangre , Ratones , Proteínas Recombinantes/inmunología , Bazo/citología , Células TH1 , Células Th2/inmunología , Vacunación , Vacunas de Subunidad/química , Vacunas de Subunidad/inmunologíaRESUMEN
BACKGROUND: Basic fibroblast growth factor (bFGF) is a member of a highly conserved superfamily of proteins that are involved in cell proliferation, differentiation, and migration. OBJECTIVES: The objective of this study was to overexpress and purify the high-level active human bFGF in Escherichia coli (E. coli). MATERIALS AND METHODS: This experimental study was conducted in the Islamic Republic of Iran. After codon optimization and gene synthesis, the optimized FGF-2 gene was subcloned into plasmid pET-32a. pET32-FGF-2 was transformed into E. coli BL21 for expression. The cultivation parameters were optimized to produce a high yield of FGF-2. RESULTS: The optimal conditions were determined as follows: cultivation at 37°C in TB medium, with 1 mM isopropyl-ß-D-thiogalactopyranoside (IPTG), followed by post-induction expression for 6 h. Under the abovementioned conditions, the expression volumetric productivity of FGF-2 reached 1.48 g/L. CONCLUSIONS: A fusion tag from the pET32 expression plasmid permits the recovery of the recombinant fusion FGF-2 from E. coli, without affecting its biological activity.
RESUMEN
PURPOSE: Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system. It has been shown that memory deficits is common in patients with MS. Recent studies using experimental autoimmune encephalomyelitis (EAE) as an animal model of MS have shown that indicated that EAE causes hippocampal-dependent impairment in learning and memory. Thus far, there have been no in vivo electrophysiological reports describing synaptic transmission in EAE animals. The aim of the present work is to evaluate the synaptic changes in the CA1 region of the hippocampus of EAE rats. METHODS: To evaluate changes in synaptic transmission in the CA1 region of the hippocampus of EAE rats, field excitatory postsynaptic potentials (fEPSPs) from the stratum radiatum of CA1 neurons, were recorded following Schaffer collateral stimulation. RESULTS: The results showed that EAE causes deficits in synaptic transmission and long-term potentiation (LTP) in the hippocampus. In addition, paired-pulse index with a 120 msec interstimulus interval was decreased in the EAE group. These findings indicate that EAE might induce suppression in synaptic transmission and LTP by increasing the inhibitory effect of GABAB receptors on the glutamate-mediated EPSP. CONCLUSIONS: In conclusion, influence of inflammation-triggered mechanisms on synaptic transmission may explain the negative effect of EAE on learning abilities in rats.
RESUMEN
Heparin-binding epidermal growth factor (HB-EGF) is a member of highly conserved superfamily of proteins that has potential mitogenic activity and stimulates differentiation and migration of various cell types. Since HB-EGF has three intra-molecular disulfide bonds, a high expression pattern of active HB-EGF in an E. coli expression system was not successfully established. The aim of this study was to increase production of soluble bioactive recombinant human HB-EGF in E. coli by modifying growth conditions and codon optimization. The open reading frame codons of human HB-EGF were optimized to achieve high level expression in E. coli. The optimized codon was amplified, cloned into plasmid pET-32a, and transformed into E. coli BL21 for further expression. The cultivation parameters (temperature and inducer) were optimized to produce a high yield of soluble HB-EGF. The fusion protein was purified by Nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. Amethylthiazole tetrazolium assay was used to evaluate the bioactivity of the produced recombinant protein. After codon optimization, the codon adaptation index (CAI) was increased from 0.255 in native gene to 0.829 using the optimized sequence. By lowering the temperature to 22°C and the inducer to 0.4 µM, we obtained 35% soluble expression of recombinant and biologically active human HB-EGF. Our data demonstrate that codon optimization increases the yield of HB-EGF in an E. coli expression system. Furthermore, the chosen modifications in cell culturing increase the solubility of recombinant human HB-EGF.