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Natural polymers have been widely used in scaffolds for tissue engineering due to their superior biocompatibility, biodegradability, and low cytotoxicity compared to synthetic polymers. Despite these advantages, there remain drawbacks such as unsatisfying mechanical properties or low processability, which hinder natural tissue substitution. Several non-covalent or covalent crosslinking methods induced by chemicals, temperatures, pH, or light sources have been suggested to overcome these limitations. Among them, light-assisted crosslinking has been considered as a promising strategy for fabricating microstructures of scaffolds. This is due to the merits of non-invasiveness, relatively high crosslinking efficiency via light penetration, and easily controllable parameters, including light intensity or exposure time. This review focuses on photo-reactive moieties and their reaction mechanisms, which are widely exploited along with natural polymer and its tissue engineering applications.
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BACKGROUND/AIMS: We aimed to assess the role of vitamin D supplementation in the response to pegylated interferon-α (PEG-IFN-α) plus ribavirin (RBV) treatment in patients with chronic hepatitis C (CHC). METHODS: Our study was a multi-center, randomized controlled trial in 11 hospitals. CHC patients were randomly assigned (1:1) to two groups namely, PEGIFN-α plus RBV (control group) or PEG-IFN-α plus RBV + vitamin D (800 IU daily) (vitamin D group). The primary end-point was the rate of sustained virologic response (SVR). RESULTS: One hundred forty eight CHC patients were randomly assigned to two groups. Seventy-one patients received the PEG-IFN-α plus RBV and 77 patients received the PEG-IFN-α plus RBV + vitamin D. A total of 105 patients completed the study (control group, 47 vs. vitamin D group, 58). Baseline characteristics were mostly similar in both the groups. There was a modest but non-significant increase in SVR in the vitamin D group compared to the control group with the intention to treat analysis (64.0% vs. 49.3 %, p = 0.071) as well as in the per protocol analysis (control group vs. vitamin D group: 74.5% vs. 84.5%, p = 0.202). Fifty-two patients (73.2%) in the control group and 63 patients (81.8%) in the vitamin D group experienced at least one adverse event. The drop-out rate due to adverse effects was not different between both groups (control group vs. vitamin D group: 19.7% vs. 10.4%, p = 0.111). CONCLUSION: Vitamin D supplement did not increase SVR in treatment naïve patients with CHC irrespective of genotype.
Assuntos
Hepatite C Crônica , Antivirais/efeitos adversos , Suplementos Nutricionais , Quimioterapia Combinada , Genótipo , Hepacivirus/genética , Hepatite C Crônica/diagnóstico , Hepatite C Crônica/tratamento farmacológico , Humanos , Polietilenoglicóis/efeitos adversos , Proteínas Recombinantes/uso terapêutico , Ribavirina/efeitos adversos , Carga Viral , Vitamina D/efeitos adversosRESUMO
BACKGROUND: Until now, various types of combined therapy with nucleotide analogs and pegylated interferon (Peg-INF) in patients with hepatitis B patients have been tried. However, studies regarding the benefits of de novo combination, late-add on, and sequential treatment are very limited. The objective of the current study was to identify the efficacy of sequential treatment of Peg-INF after short-term antiviral treatment. METHODS: Between June 2010 and June 2015, hepatitis B e antigen (HBeAg)-positive patients (n = 162) received Peg-IFN for 48 weeks (mono-treatment group, n = 81) and entecavir (ETV) for 12 weeks with a 48-week course of Peg-IFN starting at week 5 of ETV therapy (sequential treatment group, n = 81). The primary endpoint was HBeAg seroconversion at the end of follow-up period after the 24-week treatment. The primary endpoint was analyzed using Chi-square test, Fisher's exact test, and regression analysis. RESULTS: HBeAg seroconversion rate (18.2% vs. 18.2%, t = 0.03, P = 1.000) and seroclearance rate (19.7% vs. 19.7%, t = 0.03, P = 1.000) were same in both mono-treatment and sequential treatment groups. The rate of alanine aminotransferase (ALT) normalization (45.5% vs. 54.5%, t = 1.12, P = 0.296) and serum hepatitis B virus (HBV)-DNA <2000 U/L (28.8% vs. 28.8%, t = 0.10, P = 1.000) was not different in sequential and mono-treatment groups at 24 weeks of Peg-INF. Viral response rate (HBeAg seroconversion and serum HBV-DNA <2000 U/L) was not different in the two groups (12.1% vs. 16.7%, t = 1.83, P = 0.457). Baseline HBV-DNA level (7 log10U/ml vs. 7.5 log10U/ml, t = 1.70, P = 0.019) and hepatitis B surface antigen titer (3.6 log10U/ml vs. 4.0 log10U/ml, t = 2.19, P = 0.020) were lower and predictors of responder in mono-treatment and sequential treatment groups, respectively. CONCLUSIONS: The current study shows no differences in HBeAg seroconversion rate, ALT normalization, and HBV-DNA levels between mono-therapy and sequential therapy regimens. TRIAL REGISTRATION: ClinicalTrials.gov, NCT01220596; https://clinicaltrials.gov/ct2/show/NCT01220596?term=NCT01220596&rank=1.
Assuntos
Antivirais/uso terapêutico , Guanina/análogos & derivados , Guanina/uso terapêutico , Hepatite B/tratamento farmacológico , Interferon-alfa/uso terapêutico , DNA Viral , Antígenos E da Hepatite B , Hepatite B Crônica , Humanos , Polietilenoglicóis , Proteínas Recombinantes , República da Coreia , Resultado do TratamentoRESUMO
Quercetin (QT) and taxifolin (TF) are structurally similar plant-derived flavonoids that have antioxidant properties and act as free radical scavengers. The objective of this study was to investigate effects of QT and TF on nuclear maturation of porcine oocytes. Effects of TF at 0, 1, 10, and 50 µg/mL on oocyte nuclear maturation (polar body extrusion) were investigated. After incubation for 44 h, there were no significant differences between the treatment and control groups except in the 50 µg/mL group which was significantly lower (59.2%, p<0.05) than the other groups (control: >80%). After parthenogenetic activation, further in vitro development of QT- or TF-treated vs control oocytes was investigated. A significantly higher proportion of QT-treated (1 µg/mL) oocytes developed into blastocysts compared to controls (24.3% vs 16.8%, respectively); however, cleavage rate and blastocyst cell number were not affected. The TF-treated group was not significantly different from controls. Levels of reactive oxygen species (ROS) and intracellular glutathione (GSH) in oocytes and embryos in a culture medium supplemented with QT or TF were measured. Both treatment groups had significantly lower (p<0.05) levels of ROS than controls, however GSH levels were different only in QT-treated oocytes. We conclude that exogenous flavonoids such as QT and TF reduce ROS levels in oocytes. Although at high concentration (50 µg/mL) both QT and TF appear to be toxic to oocytes.
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Experimental autoimmune uveoretinitis (EAU) is an autoimmune disease that models human uveitis. Caffeic acid phenethyl ester (CAPE), a phenolic compound isolated from propolis, possesses anti-inflammatory and immunomodulatory properties. CAPE demonstrates therapeutic potential in several animal disease models through its ability to inhibit NF-κB activity. To evaluate these therapeutic effects in EAU, we administered CAPE in a model of EAU that develops after immunization with interphotoreceptor retinal-binding protein (IRBP) in B10.RIII and C57BL/6 mice. Importantly, we found that CAPE lessened the severity of EAU symptoms in both mouse strains. Notably, treated mice exhibited a decrease in the ocular infiltration of immune cell populations into the retina; reduced TNF-α, IL-6, and IFN-γ serum levels: and inhibited TNF-α mRNA expression in retinal tissues. Although CAPE failed to inhibit IRBP-specific T cell proliferation, it was sufficient to suppress cytokine, chemokine, and IRBP-specific antibody production. In addition, retinal tissues isolated from CAPE-treated EAU mice revealed a decrease in NF-κB p65 and phospho-IκBα. The data identify CAPE as a potential therapeutic agent for autoimmune uveitis that acts by inhibiting cellular infiltration into the retina, reducing the levels of pro-inflammatory cytokines, chemokine, and IRBP-specific antibody and blocking NF-κB pathway activation.
Assuntos
Doenças Autoimunes/tratamento farmacológico , Ácidos Cafeicos/uso terapêutico , Modelos Animais de Doenças , NF-kappa B/antagonistas & inibidores , Álcool Feniletílico/análogos & derivados , Retinite/tratamento farmacológico , Uveíte/tratamento farmacológico , Animais , Doenças Autoimunes/metabolismo , Doenças Autoimunes/patologia , Western Blotting , Proteínas do Olho/imunologia , Citometria de Fluxo , Imunoglobulina G/sangue , Interferon gama/sangue , Interleucina-6/metabolismo , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Álcool Feniletílico/uso terapêutico , RNA Mensageiro/metabolismo , Retinite/metabolismo , Retinite/patologia , Proteínas de Ligação ao Retinol/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/imunologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Uveíte/metabolismo , Uveíte/patologiaRESUMO
Somatic cell nuclear transfer (SCNT) is a cost-effective technique for producing transgenic pigs. However, abnormalities in the cloned pigs might prevent use these animals for clinical applications or disease modeling. In the present study, we generated several cloned pigs. One of the pigs was found to have intrapancreatic ectopic splenic tissue during histopathology analysis although this animal was grossly normal and genetically identical to the other cloned pigs. Ectopic splenic tissue in the pancreas is very rare, especially in animals. To the best of our knowledge, this is the first such report for cloned pigs.
Assuntos
Coristoma/veterinária , Técnicas de Transferência Nuclear/veterinária , Pâncreas , Esplenopatias/veterinária , Doenças dos Suínos/patologia , Animais , Animais Geneticamente Modificados , Coristoma/patologia , Clonagem de Organismos , Esplenopatias/patologia , Suínos , Porco MiniaturaRESUMO
Octamer-binding transcription factor 4 (Oct4) is a critical molecule for the self-renewal and pluripotency of embryonic stem cells. Recent reports have shown that Oct4 also controls cell-cycle progression and enhances the proliferation of various types of cells. As the high proliferation of donor fibroblasts is critical to the production of transgenic pigs, using the somatic cell nuclear transfer technique, we analysed the effect of Oct4 overexpression on the proliferation of porcine fibroblasts and embryos. Porcine endogenous Oct4 cDNA was cloned, sequenced and inserted into an expression vector. The vector was transfected into porcine fibroblasts, and a stable Oct4-overexpressed cell line was established by antibiotic selection. Oct4 expression was validated by the immunostaining of Oct4. Cell morphology was changed to sharp, and both proliferation and migration abilities were enhanced in Oct4-overexpressed cells. Real-time RT-PCR results showed that p16, Bcl2 and Myc were upregulated in Oct4-overexpressed cells. Somatic cell nuclear transfer was performed using Oct4-overexpressed cells, and the development of Oct4 embryos was compared with that of wild-type cloned embryos. The cleavage and blastocyst formation rates were improved in the Oct4 embryos. Interestingly, blastocyst formation of the Oct4 embryos was observed as early as day 5 in culture, while blastocysts were observed from day 6 in wild-type cloned embryos. In conclusion, the overexpression of Oct4 enhanced the proliferation of both porcine fibroblasts and embryos.
Assuntos
Blastocisto/citologia , Proliferação de Células , Clonagem de Organismos/métodos , Embrião de Mamíferos/citologia , Fibroblastos/citologia , Regulação da Expressão Gênica no Desenvolvimento , Fator 3 de Transcrição de Octâmero/metabolismo , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/metabolismo , Animais Recém-Nascidos , Blastocisto/metabolismo , Células Cultivadas , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário , Fibroblastos/metabolismo , Técnicas Imunoenzimáticas , Técnicas de Transferência Nuclear , Fator 3 de Transcrição de Octâmero/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Pele/citologia , Pele/metabolismo , SuínosRESUMO
Receptor activator of nuclear factor-kappa B ligand (RANKL) is a critical factor in osteoclastogenesis. It makes osteoclasts differentiate and multinucleate in bone remodeling. In the present study, RANKL was expressed as a soluble maltose binding protein (MBP)-fusion protein using the Escherichia coli maltose binding domain tag system (pMAL) expression vector system. The host cell E. coli DH5α was cultured and induced by isopropyl ß-D-1- thiogalactopyranoside for rRANKL expression. Cells were disrupted by sonication to collect soluble MBP-fused rRANKL. The MBP-fusion rRANKL was purified with MBP Trap affinity chromatography and treated with Tobacco Etch Virus nuclear inclusion endopeptidase (TEV protease) to remove the MBP fusion protein. Dialysis was then carried out to remove binding maltose from the cleaved rRANKL solution. The cleaved rRANKL was purified with a second MBP Trap affinity chromatography to separate unsevered MBP-fusion rRANKL and cleaved MBP fusion protein. The purified rRANKL was shown to have biological activity by performing in vitro cell tests. In conclusion, biologically active rRANKL was successfully purified by a simple two-step chromatography purification process with one column.
Assuntos
Escherichia coli/genética , Ligante RANK/genética , Ligante RANK/isolamento & purificação , Cromatografia de Afinidade , Endopeptidases/metabolismo , Macrófagos/fisiologia , Proteínas Ligantes de Maltose/genética , Ligante RANK/metabolismo , Proteínas Recombinantes de Fusão/isolamento & purificaçãoRESUMO
To facilitate the construction of genetically-modified pigs, we produced cloned embryos derived from porcine fibroblasts transfected with a pair of engineered zinc finger nuclease (ZFN) plasmids to create targeted mutations and enriched using a reporter plasmid system. The reporter expresses RFP and eGFP simultaneously when ZFN-mediated site-specific mutations occur. Thus, double positive cells (RFP(+)/eGFP(+)) were selected and used for somatic cell nuclear transfer. Two types of reporter based enrichment systems were used in this study; the cloned embryos derived from cells enriched using a magnetic sorting-based system showed better developmental competence than did those derived from cells enriched by flow cytometry. Mutated sequences, such as insertions, deletions, or substitutions, together with the wild-type sequence, were found in the cloned porcine blastocysts. Therefore, genetic mutations can be achieved in cloned porcine embryos reconstructed with ZFN-treated cells that were enriched by a reporter-based system.
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A Gram-negative, short-rod-shaped bacterial strain with gliding motility, designated as DG5A(T), was isolated from a rice field soil in South Korea. Phylogenic analysis using 16S rRNA gene sequence of the new isolate showed that strain DG5A(T) belong to the genus Spirosoma in the family Spirosomaceae, and the highest sequence similarities were 95.5 % with Spirosoma linguale DSM 74(T), 93.4 % with Spirosoma rigui WPCB118(T), 92.8 % with Spirosoma luteum SPM-10(T), 92.7 % with Spirosoma spitsbergense SPM-9(T), and 91.9 % with Spirosoma panaciterrae Gsoil 1519(T). Strain DG5A(T) revealed resistance to gamma and UV radiation. Chemotaxonomic data showed that the most abundant fatty acids were summed feature C(16:1) ω7c/C(16:1) ω6c (36.90 %), C(16:1) ω5c (29.55 %), and iso-C(15:0) (14.78 %), and the major polar lipid was phosphatidylethanolamine (PE). The DNA G+C content of strain DG5A(T) was 49.1 mol%. Together, the phenotypic, phylogenetic, and chemotaxonomic data supported that strain DG5A(T) presents a novel species of the genus Spirosoma, for which the name Spirosoma radiotolerans sp. nov., is proposed. The type strain is DG5A(T) (=KCTC 32455(T) = JCM19447(T)).
Assuntos
Cytophagaceae/classificação , Cytophagaceae/isolamento & purificação , Raios gama , Viabilidade Microbiana/efeitos da radiação , Microbiologia do Solo , Técnicas de Tipagem Bacteriana , Composição de Bases , Análise por Conglomerados , Cytophagaceae/fisiologia , Cytophagaceae/efeitos da radiação , Citosol/química , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Ácidos Graxos/análise , Coreia (Geográfico) , Locomoção , Microscopia Eletrônica de Transmissão , Dados de Sequência Molecular , Oryza , Fosfolipídeos/análise , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Raios UltravioletaRESUMO
Generation of transgenic pigs for xenotransplantation is one of the most promising technologies for resolving organ shortages. Human heme oxygenase-1 (hHO-1/HMOX1) can protect transplanted organs by its strong anti-oxidative, anti-apoptotic, and anti-inflammatory effects. Soluble human TNFRI-Fc (shTNFRI-Fc) can inhibit the binding of human TNF-α (hTNF-α) to TNF receptors on porcine cells, and thereby, prevent hTNF-α-mediated inflammation and apoptosis. Herein, we successfully generated shTNFRI-Fc-F2A-HA-hHO-1 transgenic (TG) pigs expressing both shTNFRI-Fc and hemagglutinin-tagged-human heme oxygenase-1 (HA-hHO-1) by using an F2A self-cleaving peptide. shTNFRI-Fc and HA-hHO-1 transgenes containing the F2A peptide were constructed under the control of the CAG promoter. Transgene insertion and copy number in the genome of transgenic pigs was confirmed by polymerase chain reaction (PCR) and Southern blot analysis. Expressions of shTNFRI-Fc and HA-hHO-1 in TG pigs were confirmed using PCR, RT-PCR, western blot, ELISA, and immunohistochemistry. shTNFRI-Fc and HA-hHO-1 were expressed in various organs, including the heart, lung, and spleen. ELISA assays detected shTNFRI-Fc in the sera of TG pigs. For functional analysis, fibroblasts isolated from a shTNFRI-Fc-F2A-HA-hHO-1 TG pig (i.e., #14; 1 × 10(5) cells) were cultured with hTNF-α (20 ng/mL) and cycloheximide (10 µg/mL). The viability of shTNFRI-Fc-F2A-HA-hHO-1 TG pig fibroblasts was significantly higher than that of the wild type (wild type vs. shTNFRI-Fc-F2A-HA-hHO-1 TG at 24 h, 31.6 ± 3.2 vs. 60.4 ± 8.3 %, respectively; p < 0.05). Caspase-3/-7 activity of the shTNFRI-Fc-F2A-HA-hHO-1 TG pig fibroblasts was lower than that of the wild type pig fibroblasts (wild type vs. shTNFRI-Fc-F2A-HA-hHO-1 TG at 12 h, 812,452 ± 113,078 RLU vs. 88,240 ± 10,438 RLU, respectively; p < 0.05). These results show that shTNFRI-Fc and HA-hHO-1 TG pigs generated by the F2A self-cleaving peptide express both shTNFRI-Fc and HA-hHO-1 molecules, which provides protection against oxidative and inflammatory injury. Utilization of the F2A self-cleaving peptide is a promising tool for generating multiple TG pigs for xenotransplantation.
Assuntos
Animais Geneticamente Modificados , Heme Oxigenase-1/biossíntese , Peptídeos/genética , Receptores Tipo I de Fatores de Necrose Tumoral/biossíntese , Animais , Apoptose/genética , Células Endoteliais/metabolismo , Fibroblastos/metabolismo , Heme Oxigenase-1/genética , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Sus scrofa , Suínos/genéticaRESUMO
The level of P4 at the time of embryo transfer (ET) is important. P4 concentrations and numbers of corpora lutea for 126 recipients were evaluated. Nuclear transfer embryos were transferred into 126 surrogates. 11 maintained their pregnancy until full-term delivery, 17 miscarried, and implantation failed in 98 animals. P4 levels in the full-term group were significantly different from those of the pigs that aborted or in which implantation failed (p < 0.05). However, the numbers of corpora lutea were not significantly different. These findings indicate that the concentration of progesterone can be an important factor for successful ET in pigs.
Assuntos
Corpo Lúteo/fisiologia , Transferência Embrionária/veterinária , Embrião de Mamíferos/fisiologia , Taxa de Gravidez , Progesterona/sangue , Sus scrofa/fisiologia , Animais , Feminino , Técnicas de Transferência Nuclear , Gravidez , Estudos RetrospectivosRESUMO
The presence of glutamine (Gln) in in vitro maturation (IVM) and in vitro culture (IVC) medium is a more potent factor for improving porcine oocyte and embryo development than other amino acids. However Gln is inherently unstable and spontaneously breaks down into ammonia, and therefore interferes with proper development. To avoid this adverse effect, Gln was replaced in the present study with its stable dipeptide derivative alanyl-glutamine (Ala-Gln) and the effects of this replacement on porcine IVM and IVC were evaluated. Replacement of Gln with Ala-Gln during IVM did not improve nuclear maturation, however numbers of early cleaved embryos were significantly increased after activation. Blastocyst formation rates were also significantly improved by using Ala-Gln during IVM. Replacement of Gln with Ala-Gln during IVC significantly increased total cell numbers in blastocysts. Blastocyst formation rate was also significantly higher when Ala-Gln was used in both IVM and IVC. In conclusion, the use of Ala-Gln rather than Gln gives better results for development in both porcine IVM and IVC.
Assuntos
Blastocisto/citologia , Dipeptídeos/farmacologia , Desenvolvimento Embrionário/efeitos dos fármacos , Fertilização in vitro , Glutamina/farmacologia , Oócitos/citologia , Animais , Blastocisto/efeitos dos fármacos , Blastocisto/fisiologia , Células Cultivadas , Fase de Clivagem do Zigoto , Técnicas de Cultura Embrionária , Feminino , Técnicas In Vitro , Oócitos/efeitos dos fármacos , Oócitos/fisiologia , SuínosRESUMO
In pig-to-primate xenotransplantation, multiple transgenic pigs are required to overcome a series of transplant rejections. The generation of multiple transgenic pigs either by breeding or the introduction of several mono-cistronic vectors has been hampered by the differential expression patterns of the target genes. To achieve simultaneous expression of multiple genes, a poly-cistronic expression system using the 2A peptide derived from the Thosea asigna virus (T2A) can be considered an alternative choice. Before applying T2A expression system to pig generation, the expression patterns of multiple genes in this system should be precisely evaluated. In this study, we constructed several bi-cistronic T2A expression vectors, which combine target genes that are frequently used in the xenotransplantation field, and introduced them into porcine fibroblasts. The proteins targeted to the same or different subcellular regions were efficiently expressed without affecting the localization or expression levels of the other protein. However, when a gene with low expression efficiency was inserted into the upstream region of the T2A sequences, the expression level of the downstream gene was significantly decreased compared with the expression efficiency without the insertion. A small interfering RNA targeting one gene in this system resulted in the significant downregulation of both the target gene and the other gene, indicating that multiple genes combined into a T2A expression vector can be considered as a single gene in terms of transcription and translation. In summary, the efficient expression of a downstream gene can be achieved if the expression of the upstream gene is efficient.
Assuntos
Cisteína Endopeptidases/genética , Fibroblastos/metabolismo , Expressão Gênica , Vetores Genéticos/genética , Animais , Linhagem Celular , Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Regulação da Expressão Gênica , Ordem dos Genes , Espaço Intracelular , Transporte Proteico , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Suínos , Transplante Heterólogo , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
One of the factors that impairs in vitro produced porcine embryos is the oxidative stress that is mainly caused by the imbalance between reactive oxygen species (ROS) generation and antioxidants activity, especially that of glutathione (GSH). Here, we examined the effect of 7,8-dihydroxyflavone (7,8-DHF), a kind of flavonoid antioxidant, on porcine oocyte maturation and its developmental competence. Porcine oocytes were cultured in media supplemented with 0, 1, 5 and 10 µM 7,8-DHF during both in vitro maturation (IVM) and in vitro culture (IVC) after parthenogenetic activation. Maturation of oocytes was evaluated based on first polar body (PB) extrusion and intracellular GSH level, and developmental competence was assessed through observing cleavage and blastocyst formation. In each step, the levels of intracellular GSH and ROS were assessed by fluorescence intensity, and the apoptosis-related gene expression was examined using semiquantitative RT-PCR. The group treated with 1 µM 7,8-DHF during IVM and IVC showed increased cytoplasmic maturation and reached the blastocysts stage (36.1%) at a higher rate than the other groups (24.7, 16.0 and 10.3% for 0, 5 and 10 µM, P<0.05). In that group, the intracellular GSH level was significantly increased while ROS generation was significantly decreased after IVM and IVC (P<0.05). Moreover, it showed high expression of an anti-apoptotic gene (BCL2L1) and low expression of a pro-apoptotic gene (BAK1) (P<0.05). In conclusion, treatment with 1 µM 7,8-DHF during IVM and IVC showed an anti-apoptotic effect by increasing intracellular GSH synthesis and scavenging ROS and therefore improved the developmental competence of porcine embryos.
Assuntos
Antioxidantes/farmacologia , Blastocisto/efeitos dos fármacos , Ectogênese/efeitos dos fármacos , Flavonas/farmacologia , Oócitos/efeitos dos fármacos , Partenogênese/efeitos dos fármacos , Sus scrofa , Matadouros , Animais , Antioxidantes/efeitos adversos , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Blastocisto/metabolismo , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Células Cultivadas , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Regulação para Baixo/efeitos dos fármacos , Técnicas de Cultura Embrionária/veterinária , Feminino , Flavonas/efeitos adversos , Glutationa/agonistas , Glutationa/metabolismo , Técnicas de Maturação in Vitro de Oócitos/veterinária , Oócitos/citologia , Oócitos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
Quercetin is a plant-derived flavonoid found in fruits or vegetables that has antioxidant properties and acts as a free radical scavenger. We investigated the effects of quercetin on porcine oocyte nuclear maturation and embryonic development after parthenogenetic activation. We then evaluated the antioxidant activities of quercetin by measuring reactive oxygen species (ROS) levels in matured oocytes. Immature oocytes were untreated or treated with 1, 10, and 50 µg/mL quercetin during in vitro maturation (IVM). Quercetin treatment did not improve oocyte nuclear maturation, but significantly higher blastocyst rates (p < 0.05) of parthenogenetically activated oocytes were achieved when the IVM medium was supplemented with an adequate concentration of quercetin (1 µg/mL). However, cleavage rates and blastocyst cell numbers were not affected. Oocytes treated with 1 or 10 µg/mL quercetin had significantly lower (p < 0.05) levels of ROS than the control and group treated with the highest concentration of quercetin (50 µg/mL). Moreover, this highest concentration was detrimental to oocyte nuclear maturation and blastocyst formation. Based on our findings, we concluded that exogenous quercetin reduces ROS levels during oocyte maturation and is beneficial for subsequent embryo development.
Assuntos
Antioxidantes/farmacologia , Técnicas de Maturação in Vitro de Oócitos/veterinária , Oócitos/efeitos dos fármacos , Quercetina/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Suínos , Animais , Antioxidantes/administração & dosagem , Relação Dose-Resposta a Droga , Oócitos/citologia , Oócitos/fisiologia , Quercetina/administração & dosagemRESUMO
Xenotransplantation using transgenic pigs as an organ source is a promising strategy to overcome shortage of human organ for transplantation. Various genetic modifications have been tried to ameliorate xenograft rejection. In the present study we assessed effect of transgenic expression of human heme oxygenase-1 (hHO-1), an inducible protein capable of cytoprotection by scavenging reactive oxygen species and preventing apoptosis caused by cellular stress during inflammatory processes, in neonatal porcine islet-like cluster cells (NPCCs). Transduction of NPCCs with adenovirus containing hHO-1 gene significantly reduced apoptosis compared with the GFP-expressing adenovirus control after treatment with either hydrogen peroxide or hTNF-α and cycloheximide. These protective effects were diminished by co-treatment of hHO-1 antagonist, Zinc protoporphyrin IX. We also generated transgenic pigs expressing hHO-1 and analyzed expression and function of the transgene. Human HO-1 was expressed in most tissues, including the heart, kidney, lung, pancreas, spleen and skin, however, expression levels and patterns of the hHO-1 gene are not consistent in each organ. We isolate fibroblast from transgenic pigs to analyze protective effect of the hHO-1. As expected, fibroblasts derived from the hHO-1 transgenic pigs were significantly resistant to both hydrogen peroxide damage and hTNF-α and cycloheximide-mediated apoptosis when compared with wild-type fibroblasts. Furthermore, induction of RANTES in response to hTNF-α or LPS was significantly decreased in fibroblasts obtained from the hHO-1 transgenic pigs. These findings suggest that transgenic expression of hHO-1 can protect xenografts when exposed to oxidative stresses, especially from ischemia/reperfusion injury, and/or acute rejection mediated by cytokines. Accordingly, hHO-1 could be an important candidate molecule in a multi-transgenic pig strategy for xenotransplantation.
Assuntos
Animais Geneticamente Modificados/genética , Heme Oxigenase-1/genética , Sus scrofa/genética , Adenoviridae/genética , Animais , Apoptose , Sobrevivência Celular , Células Cultivadas , Citocinas/fisiologia , Feminino , Fibroblastos/enzimologia , Fibroblastos/imunologia , Fibroblastos/fisiologia , Engenharia Genética , Heme Oxigenase-1/biossíntese , Heme Oxigenase-1/fisiologia , Humanos , Ilhotas Pancreáticas/enzimologia , Ilhotas Pancreáticas/fisiologia , Lipopolissacarídeos/farmacologia , Masculino , Especificidade de Órgãos , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Transplante Heterólogo , Fator de Necrose Tumoral alfa/farmacologia , Fator de Necrose Tumoral alfa/fisiologiaRESUMO
Abstract Aberrant epigenetic nuclear reprogramming of somatic nuclei is a major cause of low success in cloning. It has been demonstrated that treatment of histone deacetylase inhibitors (HDACi) enhances developmental potential of somatic cell nuclear transfer (SCNT) embryos by alteration of epigenetic status. The aim of the present study was to investigate the effect of oxamflatin, a novel HDACi, on the developmental competence of porcine SCNT embryos. Treatment with 1 µM oxamflatin for 9 h after activation of SCNT embryos increased both in vitro and in vivo developmental competence. Treatment of SCNT embryos with 1 µM oxamflatin significantly increased blastocyst rate and total cell number in blastocysts (33.3±6.0 and 73.1±1.6, respectively) than that of controls (10.3±3.7 and 54.1±3.5, respectively) or scriptaid (16.4±4.6 and 64.4±2.1, respectively). Moreover, oxamflatin showed significant higher overall cloning efficiency from 0.9% to 3.2%, whereas scriptaid demonstrated 0% to 1.8%. In conclusion, these results indicate that oxamflatin treatment improves the developmental competence of porcine SCNT embryos.
Assuntos
Transferência Embrionária , Ácidos Hidroxâmicos/farmacologia , Técnicas de Transferência Nuclear , Animais , Feminino , Inibidores de Histona Desacetilases/farmacologia , Hidroxilaminas/farmacologia , Partenogênese , Gravidez , Quinolinas/farmacologia , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , SuínosRESUMO
Kisspeptin (Kp) is best known as a multifunctional peptide with roles in reproduction, the cardiovascular system and cancer. In the present study the expression of kisspeptin hierarchy elements (KISS1, GNRH1 and LHB) and their receptors (KISS1R, GNRHR and LHCGR, respectively) in porcine ovary and in cumulus-oocyte complexes (COCs) were investigated, as were its effects on the in vitro maturation (IVM) of oocytes and their subsequent ability to sustain preimplantation embryo competence after parthenogenetic electrical activation. Kp system elements were expressed and affected IVM of oocytes when maturation medium was supplemented with 10(-6)M Kp. Oocyte maturation, maternal gene expression (MOS, GDF9 and BMP15), blastocyst formation rate, blastocyst hatching and blastocyst total cell count were all significantly increased when oocytes were matured in medium containing Kp compared with the control group (without Kp). A Kp antagonist (p234) at 4×10(-6)M interfered with this hierarchy but did not influence the threshold effect of gonadotrophins on oocyte maturation. FSH was critical and permissive to Kp action on COCs by increasing the relative expression of KISS1R. In contrast, Kp significantly increased apoptosis, the expression of pro-apoptotic gene, BAK1, and suppressed trophoblast outgrowths from hatched blastocysts cultured on feeder cells. The present study provides the first functional evidence of the Kp hierarchy in porcine COCs and its role in enhancing oocyte maturation and subsequent developmental competence in an autocrine-paracrine manner. However, Kp supplementation may have a harmful impact on cultured hatched blastocysts reflecting systemic or local regulation during the critical early period of embryonic development.
Assuntos
Blastocisto/efeitos dos fármacos , Técnicas de Maturação in Vitro de Oócitos , Kisspeptinas/farmacologia , Oócitos/efeitos dos fármacos , Animais , Blastocisto/metabolismo , Blastocisto/fisiologia , Células Cultivadas , Técnicas de Cultura Embrionária , Desenvolvimento Embrionário/efeitos dos fármacos , Desenvolvimento Embrionário/genética , Desenvolvimento Embrionário/fisiologia , Feminino , Técnicas de Maturação in Vitro de Oócitos/veterinária , Kisspeptinas/genética , Kisspeptinas/metabolismo , Oócitos/metabolismo , Oócitos/fisiologia , Oogênese/efeitos dos fármacos , Oogênese/genética , Partenogênese/efeitos dos fármacos , Partenogênese/genética , Partenogênese/fisiologia , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , SuínosRESUMO
In this study, we investigated the effect of two oxygen concentrations (5 and 20%) during in vitro maturation (IVM) and during in vitro culture (IVC) on porcine embryo development and analysed differences in gene expression between cumulus-oocyte complexes matured under 5 or 20% oxygen and the resulting blastocysts cultured under 5% or 20% oxygen following parthenogenetic activation. There was no significant difference in oocyte maturation rate. However, the numbers of resulting blastocysts were significantly increased in the 5% IVC group compared with the 20% IVC group. Moreover, the M20C5 treatment group (23.01%) supported greater blastocyst development compared with the M5C5 (14.32%), M5C20 (10.30%), and M20C20 (17.88%) groups. However, total cell numbers were not significantly different among groups. According to mRNA abundance data of multiple genes, each treatment altered the expression of genes in different patterns. GLUT1, G6PD and LDHA were up-regulated in cumulus cells that had been matured in low oxygen, suggesting a higher glucose uptake and an increase in anaerobic glycolysis, whereas cyclin B1 (CCNB) and MnSOD (Mn-superoxide dismutase) were upregulated in cumulus cells that had been matured in high oxygen, which suggests a higher activity of mitosis-promoting factor and antioxidant response. In spite of these differential effects on cumulus cells, oocytes could mature normally regardless of different oxygen concentrations. Therefore, it can be concluded that high oxygen concentration during in vitro maturation and low oxygen during in vitro culture may alter the expression of multiple genes related to oocyte competence and significantly improves embryo development (p < 0.05) but not blastocyst quality.