RESUMO
Stem cell therapy faces many problems including poor survival rates and low viability. Enhancing the biological functions of stem cells improves efficacy of therapies. Estrogen, whose levels are elevated during pregnancy, affects the properties of bone marrow mesenchymal stem cells. Evidence suggests that adipose-derived stem cells (ADSCs), which are a type of adult mesenchymal stem cells, can be used in regenerative medicine. In fact, ADSCs from pregnant animals have been used in clinical therapies. However, the effect of the donor's reproductive status on proliferation of ADSCs is unknown. We investigated the effect of 17ß-estradiol (E2) and progesterone (P) on the in vitro proliferation of ADSCs from laboratory rats. ADSCs were obtained from five different groups of 15 rats each - non-pregnant, pregnant, in perinatal period, non-pregnant and treated with E2, and non-pregnant and treated with P. Adhesion and viability of ADSCs were determined by MTT assay, and cell cycle was followed by flow cytometry. The proliferation rate of ADSCs from pregnant rats was significantly higher than those from the non-pregnant rats (P < 0.05); however, there was no statistically significant difference in proliferation rates during different phases of pregnancy (P > 0.05). Additionally, ADSCs from pregnant rats possess higher adhesion property in early stage (P1 passage) and higher proliferation rate than ADSCs from non-pregnant rats. Interestingly, ADSCs from non-pregnant rats that were treated with E2, but not those treated with P, showed higher proliferation rates than those from their untreated counterparts. These results suggest that the proliferative capacity and residence time in different cell cycle phases of ADSCs can be regulated by extrinsic factors such as estrogen concentration.
Assuntos
Tecido Adiposo/citologia , Diferenciação Celular , Células-Tronco/citologia , Células-Tronco/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Estradiol/farmacologia , Feminino , Gravidez , Progesterona/farmacologia , Ratos , Células-Tronco/efeitos dos fármacosRESUMO
In this study, we compared the functional properties of endothelial progenitor cells (EPCs) derived from halfpipe-snowboarding athletes who train under hyperoxic conditions with those derived from normal subjects who lived under normoxic conditions. Peripheral blood-derived EPCs were isolated from both halfpipe-snowboarding athletes and normal humans. Cellular growth dynamics, lipoprotein transport, and gene expression of cultured EPCs were compared between the two groups of cells. Results indicate that cytoactivity of EPCs from athletes was higher than that of EPCs from control subjects. This study suggests that function of EPCs from snowboarding athletes may be better than that of EPCs from normal humans, which demonstrates the benefits of training under hyperoxic conditions.
Assuntos
Células Progenitoras Endoteliais/citologia , Exercício Físico , Expressão Gênica , Lipoproteínas/metabolismo , Atletas , Hipóxia Celular , Proliferação de Células , Células Cultivadas , Células Progenitoras Endoteliais/metabolismo , Humanos , EsquiRESUMO
MicroRNAs (miRs) are associated with tumor progression in various cancers, such as gastric and hepatic carcinomas, and lung cancer. miR-301a is overexpressed and displays oncogenic activity in cancers. We investigated the biological involvement of miR-301a in osteosarcoma (OS). Quantitative real-time polymerase chain reaction (qRT-PCR) was used to analyze expression levels of miR-301a in 24 OS and matched adjacent non-tumor tissues. A miR-301a mimic was transferred into OS cell lines U-2 OS and MG-63 to upregulate miR-301a. The effects of miR-301a were investigated by examining cell proliferation, migration, and the cell cycle. The miR-301 target was predicted by TargetScan and confirmed by western blotting and qRT-PCR. The expression of miR-301a was significantly higher in OS tissues compared with the matched adjacent non-tumor tissues (0.959 ± 0.39 vs 3.9516 ± 1.18). Upregulated miR-301a significantly increased proliferation at 48 and 72 h compared to the negative control (U-2 OS: 2.11 ± 0.21 vs 2.88 ± 0.24; 2.70 ± 0.26 vs 3.71 ± 0.24; MG-63: 2.19 ± 0.20 vs 3.19 ± 0.22; 3.1 ± 0.25 vs 4.01 ± 0.27) and migration capability (U-2 OS: 100 ± 20.19 vs 150.68 ± 32.83; MG-63: 100 ± 17.20 vs 133.35 ± 26.26), and decreased apoptosis in both U-2 OS (10.87 ± 2.53 vs 4.01 ± 2.23) and MG-63 (15.26 ± 2.15 vs 8.25 ± 3.07). The cell cycle studies revealed that miR-301a caused an increase of the G2 population in U-2 OS (38.6 ± 6.58 vs 47.2 ± 7.27) and MG-63 (44.01 ± 5.28 vs 57.9 ± 4.25). Additional experiments indicated that CDC14A was upregulated by miR-301a (0.63 ± 0.06 vs 0.98 ± 0.06; 1.49 ± 0.25 vs 2.99 ± 0.14). Overexpressed miR-301a may increase CDC14A expression and promote cell proliferation and migration in OS cells. Therefore, miR- 301a may be useful for osteosarcoma diagnosis and therapy.
Assuntos
Carcinogênese/genética , MicroRNAs/biossíntese , Osteossarcoma/genética , Monoéster Fosfórico Hidrolases/biossíntese , Apoptose/genética , Ciclo Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , MicroRNAs/genética , Osteossarcoma/patologia , Monoéster Fosfórico Hidrolases/genética , Proteínas Tirosina Fosfatases , Ativação Transcricional/genéticaRESUMO
As a heterogeneous group of disorders in pregnancy, many genetic factors are involved in the development of preeclampsia. The single nucleotide polymorphism (SNP) rs7579169, located on chromosome 2q14.2, has been shown to be associated with pregnancy-induced hypertension in Europeans. In this study, we examined whether the SNP rs7579169 is associated with the susceptibility to preeclampsia through a case-control research model in Han Chinese women. Genotypes of 145 patients with preeclampsia and 150 healthy pregnant subjects were identified by direct sequencing. The correlation between the rs7579169 genotype and the susceptibility to preeclampsia was evaluated using an unconditional logistic regression model. Although there were no differences of having the rs7579169 SNP between early onset and late onset preeclampsia, patients carrying the CT or TT genotype were more likely to develop preeclampsia than those carrying the CC genotype (CT vs CC: OR = 1.76, 95%CI = 1.07-2.87, P < 0.05; TT vs CC: OR = 5.03, 95%CI = 1.99-12.73, P < 0.05; CC vs CT + TT: OR = 2.05, 95%CI = 1.27-3.30, P < 0.05). In conclusion, although no differences of the rs7579169 SNP were identified between the early onset and late onset preeclampsia groups, we found that the CT or TT genotype and the CT+TT genotype were significantly associated with an increased risk of preeclampsia in Han Chinese women.
Assuntos
Povo Asiático/genética , Cromossomos Humanos Par 2 , Pré-Eclâmpsia/genética , Adulto , Alelos , Estudos de Casos e Controles , China , Etnicidade/genética , Feminino , Frequência do Gene , Predisposição Genética para Doença , Genótipo , Humanos , Hipertensão Induzida pela Gravidez/genética , Modelos Logísticos , Polimorfismo de Nucleotídeo Único , Gravidez , Fatores de RiscoRESUMO
Bone fractures or bones subjected to open conduction and internal fixation are easily infected by bacteria; bacterial lipopolysaccharide (LPS) has been recognized as an important pathogenic factor affecting bone fracture healing. Therefore, the effect of LPS on bone metabolism is relevant for bone healing. In this study, we investigated the effect of LPS on the expression of Toll-like receptor (TLR)-4 (an LPS receptor) by using real-time quantitative PCR and western blotting. We also examined the regulatory role of LPS in osteoblast differentiation by measuring the ALP activity, matrix mineralization, and ALP, OCN, and Runx2 mRNA (essential factors affecting osteoblast differentiation) expression in LPS-treated mouse osteoblast MC3T3-E1 cells. We also evaluated the effect of TLR-4 on LPS-mediated inhibition of osteoblast differentiation using RNA interference. LPS promotes TLR-4 mRNA and protein expression in MC3T3-E1 cells (P < 0.05, P < 0.01 or P < 0.001), and inhibits osteoblast differentiation by downregulating matrix mineralization and ALP activity (P < 0.05, P < 0.01 or P < 0.001), and suppressing the expression ALP, OCN, and Runx2 mRNA in MC3T3-E1 cells (P < 0.05 or P < 0.01). Conversely, RNAi-mediated TLR-4 knockdown abrogates the LPS-mediated inhibition of osteoblast differentiation (P < 0.05 or P < 0.01). In summary, LPS was shown to inhibit osteoblast differentiation by suppressing the expression of ALP, OCN, and Runx2 in a TLR-4-dependent manner. The results of this study may provide insights into the signal pathway of LPS-induced bone loss or delayed bone fracture healing.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Fraturas Ósseas/genética , Osteoblastos/metabolismo , Receptor 4 Toll-Like/biossíntese , Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Animais , Linhagem Celular , Subunidade alfa 1 de Fator de Ligação ao Core/biossíntese , Fraturas Ósseas/tratamento farmacológico , Fraturas Ósseas/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Lipopolissacarídeos/administração & dosagem , Proteínas de Membrana/biossíntese , Camundongos , Osteoblastos/patologia , Osteogênese/efeitos dos fármacos , RNA Mensageiro/genética , Transdução de Sinais/efeitos dos fármacos , Receptor 4 Toll-Like/genéticaRESUMO
The genes of top athletes are a valuable genetic resource for the human race, and could be exploited to identify novel genes related to sports ability, as well as other functions. We analyzed the expressed sequence tags from top half-pipe snowboarding athletes using the SMART complementary DNA (cDNA) library construction method to elucidate the characteristics of the athlete genome and the differential expression of the genes it contains. Overall, we established a full-length cDNA library from the lymphocytes of half-pipe snowboarding athletes and analyzed the inserted gene fragments. We also classified those genes according to molecular function, biological characteristics, cellular composition, protein types, and signal paths. A total of 201 functional genes were noted, which were distributed in 27 pathways. TXN, MDH1, ARL1, ARPC3, ACTG1, and other genes measured in sequence may be associated with physical ability. This suggests that the SMART cDNA library constructed from the genetic material from top athletes is an effective tool for preserving genetic sports resources and providing genetic markers of physical ability for athlete selection.
Assuntos
Atletas , Etiquetas de Sequências Expressas , Biblioteca Gênica , Genes/genética , Linfócitos , Esqui , Clonagem Molecular , HumanosRESUMO
FKBP38 (also known as FKBP8) is a unique member of the FK506-binding protein (FKBP) family, and its role is controversial because it acts as an upstream regulator of the mTOR signaling pathway, which controls cell growth, proliferation, and differentiation. This study aimed to explore the role of FKBP38 in the activation of mTOR signaling in Cashmere goat (Capra hircus) fetal fibroblasts. To construct a Cashmere goat FKBP38 siRNA eukaryotic expression vector that targets FKBP38 mRNA, we designed shRNA based on the gene sequence deposited in GenBank (accession No. JF714970) and synthesized a DNA fragment encoding the shRNA. The DNA fragment was inserted into the pRNAT-U6.1/Neo vector to construct an expression vector of shRNA, which was labeled pRNAT-FKBP38-shRNA. The recombinant plasmid was used to transfect Cashmere goat fetal fibroblasts (GFb) using lipofectamine™2000. We found that cells were successfully transfected with pRNAT-U6.1/Neo-FKBP38-shRNA. Green fluorescence could be observed in cells following 48-h transfection. Proteins were then isolated from GFbs transfected with pRNAT-FKBP38-shRNA and from control cells, and protein expression was analyzed by western blot. Expression of FKBP38 decreased and mTOR signaling was activated, which induced the phosphorylation of mTOR, S6, and 4EBP1. Thus, FKBP38 gene-silencing activates mTOR signaling in goat cells.
Assuntos
Fibroblastos/metabolismo , Inativação Gênica , Cabras/genética , Cabras/metabolismo , RNA Interferente Pequeno/genética , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Proteínas de Ligação a Tacrolimo/genética , Animais , Sequência de Bases , Células Cultivadas , Expressão Gênica , Ordem dos Genes , Vetores Genéticos/genética , Fosforilação , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Interferente Pequeno/química , Proteínas Quinases S6 Ribossômicas/metabolismo , Proteínas de Ligação a Tacrolimo/química , TransfecçãoRESUMO
Osteoporosis is the most common bone disease, affecting millions of people worldwide and leading to significant morbidity and high costs. Monacolin K, an extract of red yeast rice (RYR, Hongqu), plays important roles in the management of dyslipidemia, coronary heart disease, and diabetes. Our study aimed to investigate the protective effect of monacolin K on ovariectomy-induced bone loss in rats. Fifty female Sprague-Dawley rats were randomly divided into a sham-operated and five ovariectomized (OVX) groups: OVX with vehicle, OVX with fluvastatin, and OVX with RYR extract of three graded doses. Bone mineral density (BMD), biochemical markers, and cell viability were analyzed by dual energy X-ray absorptiometry, enzyme-linked immunosorbent assay, and 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays. Gene expression was evaluated by real-time polymerase chain reaction amplification and western blot. Our results showed that administration of RYR extract markedly increased the bone mineral density in OVX rats. Moreover, RYR extract decreased the levels of bone turnover markers, including osteocalcin and tartrate resistant acid phosphatase activity. The MMT assay revealed that RYR extract treatment significantly improved the osteoblast viabilities in a dose-dependent manner (P < 0.05). At the molecular level, we further demonstrated that RYR extract enhanced the expression of Bmp2 and Bmp4 both at the mRNA and protein levels. Collectively, these data suggested RYR extract could protect against osteoporosis in ovariectomized rats, most likely through activation of BMP2/4 expression.
Assuntos
Produtos Biológicos/uso terapêutico , Reabsorção Óssea/tratamento farmacológico , Reabsorção Óssea/etiologia , Osteoporose/tratamento farmacológico , Ovariectomia , Extratos Vegetais/uso terapêutico , Animais , Produtos Biológicos/farmacologia , Biomarcadores/metabolismo , Peso Corporal/efeitos dos fármacos , Densidade Óssea/efeitos dos fármacos , Proteínas Morfogenéticas Ósseas/metabolismo , Remodelação Óssea/efeitos dos fármacos , Reabsorção Óssea/complicações , Proliferação de Células/efeitos dos fármacos , Feminino , Tamanho do Órgão/efeitos dos fármacos , Osteoporose/complicações , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Útero/efeitos dos fármacos , Útero/patologiaRESUMO
Fibroblast growth factor 5 (FGF5) is a secreted signaling protein that belongs to the FGF family, and was found to be associated with hair growth in humans and other animals. The Inner Mongolia Cashmere goat (Capra hircus) is a goat breed that provides superior cashmere; this breed was formed by spontaneous mutation in China. Here, we report the cloning, molecular characterization, and expression pattern of the Cashmere goat FGF5. The cloned FGF5 cDNA was 813 base pairs (KM596772), including an open reading frame encoding a 270-amino-acid polypeptide. The nucleotide sequence shared 99% homology with Ovis aries FGF5 (NM_001246263.1). Bioinformatic analysis revealed that FGF5 contained a signal peptide, an FGF domain, and a heparin-binding growth factor/FGF family signature. There was 1 cAMP- and cGMP-dependent protein kinase phosphorylation site, 11 protein kinase C phosphorylation sites, 4 casein kinase II phosphorylation sites, 1 amidation site, 1 N-glycosylation site, and 1 tyrosine kinase phosphorylation site in FGF5. Real-time polymerase chain reaction showed that FGF5 mRNA levels were higher in testis than in the pancreas and liver. These data suggest that FGF5 may play a crucial role in Cashmere goat hair growth.
Assuntos
Fator 5 de Crescimento de Fibroblastos/genética , Expressão Gênica , Cabras/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Células Cultivadas , Clonagem Molecular , Fator 5 de Crescimento de Fibroblastos/química , Fator 5 de Crescimento de Fibroblastos/metabolismo , Cabras/genética , Masculino , Modelos Moleculares , Dados de Sequência Molecular , Especificidade de Órgãos , Filogenia , RNA Mensageiro , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Testículo/metabolismoRESUMO
Protein kinases regulate many processes, including cell growth, metabolism, molecular interactions, and cell proliferation. Protein kinase B (PKB)/AKT (v-AKT mouse thymoma viral oncogene homolog) is an upstream component of mammalian target of rapamycin (mTOR) signaling and mediates pathophysiological processes in several signaling pathways. This study aimed to construct and overexpress a eukaryotic goat AKT expression vector in goat fetal fibroblasts and examine the effects of AKT on the phosphorylation of p70S6K and 4E-BP1. AKT was subcloned into the expression vector pIRES2-DsRed2 to generate pIRES2-DsRed2-AKT, which was transfected into goat fetal fibroblasts with LipofectamineTM 2000. AKT was measured by reverse transcription-polymerase chain reaction in the transgenic cells, and the expression of AKT and phosphorylation of p70S6K (Thr389) and 4E-BP1 (Thr37/46) were analyzed by Western blot. Cell clones that stably emitted red fluorescence were obtained after transfection for 48 h, and the exogenous gene was verified. Exogenous AKT was transcribed, and AKT was overexpressed, inducing the phosphorylation of p70S6K (Thr389) and 4E-BP1 (Thr37/46) in goat fetal fibroblasts. Thus, the overexpression of AKT activates mTOR signaling in goat cells.
Assuntos
Proteínas de Transporte/metabolismo , Feto/citologia , Fibroblastos/enzimologia , Cabras/embriologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Animais , Células Clonais , Vetores Genéticos/metabolismo , Fosforilação , Recombinação Genética/genética , Transfecção , TransgenesRESUMO
People who suffer from traumatic brain injury (TBI) often experience cognitive deficits in spatial reference and working memory. The possible roles of cyclooxygenase-1 (COX-1) in learning and memory impairment in mice with TBI are far from well known. Adult mice subjected to TBI were treated with the COX-1 selective inhibitor SC560. Performance in the open field and on the beam walk was then used to assess motor and behavioral function 1, 3, 7, 14, and 21 days following injury. Acquisition of spatial learning and memory retention was assessed using the Morris water maze on day 15 post-TBI. The expressions of COX-1, prostaglandin E2 (PGE2), interleukin (IL)-6, brain-derived neurotrophic factor (BDNF), platelet-derived growth factor BB (PDGF-BB), synapsin-I, and synaptophysin were detected in TBI mice. Administration of SC560 improved performance of beam walk tasks as well as spatial learning and memory after TBI. SC560 also reduced expressions of inflammatory markers IL-6 and PGE2, and reversed the expressions of COX-1, BDNF, PDGF-BB, synapsin-I, and synaptophysin in TBI mice. The present findings demonstrated that COX-1 might play an important role in cognitive deficits after TBI and that selective COX-1 inhibition should be further investigated as a potential therapeutic approach for TBI.
Assuntos
Animais , Lesões Encefálicas/complicações , Córtex Cerebral/lesões , Ciclo-Oxigenase 1/fisiologia , Inibidores de Ciclo-Oxigenase/uso terapêutico , Aprendizagem/efeitos dos fármacos , Transtornos da Memória/tratamento farmacológico , Pirazóis/uso terapêutico , Western Blotting , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Descorticação Cerebral , Ciclo-Oxigenase 1/metabolismo , Modelos Animais de Doenças , Dinoprostona/análise , Dinoprostona/metabolismo , Ensaio de Imunoadsorção Enzimática , Hipocampo/metabolismo , /sangue , Aprendizagem em Labirinto/efeitos dos fármacos , Transtornos da Memória/etiologia , Transtornos da Memória/metabolismo , Proteínas Proto-Oncogênicas c-sis/metabolismo , Recuperação de Função Fisiológica/efeitos dos fármacos , Sinaptofisina/análise , Sinaptofisina/metabolismoRESUMO
In this study, we investigated the potential role of high-mobility group box 1 (HMGB1) in severe acute pancreatitis (SAP) and the effects of growth hormone (G) and somatostatin (S) in SAP rats. The rats were randomly divided into 6 groups of 20 each: sham-operated, SAP, SAP+saline, SAP+G, SAP+S and SAP+G+S. Ileum and pancreas tissues of rats in each group were evaluated histologically. HMGB1 mRNA expression was measured by reverse transcription-PCR. Levels of circulating TNF-α, IL-1, IL-6, and endotoxin were also measured. In the SAP group, interstitial congestion and edema, inflammatory cell infiltration, and interstitial hemorrhage occurred in ileum and pancreas tissues. The levels of HMGB1, TNF-α, IL-1, IL-6 and endotoxin were significantly up-regulated in the SAP group compared with those in the sham-operated group, and the 7-day survival rate was 0%. In the SAP+G and SAP+S groups, the inflammatory response of the morphological structures was alleviated, the levels of HMGB1, TNF-α, IL-1, IL-6, and endotoxin were significantly decreased compared with those in the SAP group, and the survival rate was increased. Moreover, in the SAP+G+S group, all histological scores were significantly improved and the survival rate was significantly higher compared with the SAP group. In conclusion, HMGB1 might participate in pancreas and ileum injury in SAP. Growth hormone and somatostatin might play a therapeutic role in the inflammatory response of SAP.
Assuntos
Animais , Masculino , Hormônio do Crescimento/metabolismo , Proteína HMGB1/metabolismo , Pâncreas/patologia , Pancreatite Necrosante Aguda/etiologia , Somatostatina/metabolismo , Edema/patologia , Endotoxinas/sangue , Expressão Gênica , Proteína HMGB1/genética , Hematoma/patologia , Íleo/lesões , Íleo/patologia , Interleucina-1beta/sangue , /sangue , Microscopia Eletrônica de Transmissão , Infiltração de Neutrófilos/fisiologia , Pâncreas/lesões , Pâncreas/metabolismo , Pancreatite Necrosante Aguda/metabolismo , Pancreatite Necrosante Aguda/patologia , Distribuição Aleatória , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , RNA Mensageiro/isolamento & purificação , Taxa de Sobrevida , Fator de Necrose Tumoral alfa/sangueRESUMO
People who suffer from traumatic brain injury (TBI) often experience cognitive deficits in spatial reference and working memory. The possible roles of cyclooxygenase-1 (COX-1) in learning and memory impairment in mice with TBI are far from well known. Adult mice subjected to TBI were treated with the COX-1 selective inhibitor SC560. Performance in the open field and on the beam walk was then used to assess motor and behavioral function 1, 3, 7, 14, and 21 days following injury. Acquisition of spatial learning and memory retention was assessed using the Morris water maze on day 15 post-TBI. The expressions of COX-1, prostaglandin E2 (PGE2), interleukin (IL)-6, brain-derived neurotrophic factor (BDNF), platelet-derived growth factor BB (PDGF-BB), synapsin-I, and synaptophysin were detected in TBI mice. Administration of SC560 improved performance of beam walk tasks as well as spatial learning and memory after TBI. SC560 also reduced expressions of inflammatory markers IL-6 and PGE2, and reversed the expressions of COX-1, BDNF, PDGF-BB, synapsin-I, and synaptophysin in TBI mice. The present findings demonstrated that COX-1 might play an important role in cognitive deficits after TBI and that selective COX-1 inhibition should be further investigated as a potential therapeutic approach for TBI.
Assuntos
Lesões Encefálicas/complicações , Córtex Cerebral/lesões , Ciclo-Oxigenase 1/fisiologia , Inibidores de Ciclo-Oxigenase/uso terapêutico , Aprendizagem/efeitos dos fármacos , Transtornos da Memória/tratamento farmacológico , Pirazóis/uso terapêutico , Animais , Becaplermina , Western Blotting , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Descorticação Cerebral , Ciclo-Oxigenase 1/metabolismo , Dinoprostona/análise , Dinoprostona/metabolismo , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Hipocampo/metabolismo , Interleucina-6/sangue , Aprendizagem em Labirinto/efeitos dos fármacos , Transtornos da Memória/etiologia , Transtornos da Memória/metabolismo , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas c-sis/metabolismo , Recuperação de Função Fisiológica/efeitos dos fármacos , Sinaptofisina/análise , Sinaptofisina/metabolismoRESUMO
In this study, we investigated the potential role of high-mobility group box 1 (HMGB1) in severe acute pancreatitis (SAP) and the effects of growth hormone (G) and somatostatin (S) in SAP rats. The rats were randomly divided into 6 groups of 20 each: sham-operated, SAP, SAP+saline, SAP+G, SAP+S and SAP+G+S. Ileum and pancreas tissues of rats in each group were evaluated histologically. HMGB1 mRNA expression was measured by reverse transcription-PCR. Levels of circulating TNF-α, IL-1, IL-6, and endotoxin were also measured. In the SAP group, interstitial congestion and edema, inflammatory cell infiltration, and interstitial hemorrhage occurred in ileum and pancreas tissues. The levels of HMGB1, TNF-α, IL-1, IL-6 and endotoxin were significantly up-regulated in the SAP group compared with those in the sham-operated group, and the 7-day survival rate was 0%. In the SAP+G and SAP+S groups, the inflammatory response of the morphological structures was alleviated, the levels of HMGB1, TNF-α, IL-1, IL-6, and endotoxin were significantly decreased compared with those in the SAP group, and the survival rate was increased. Moreover, in the SAP+G+S group, all histological scores were significantly improved and the survival rate was significantly higher compared with the SAP group. In conclusion, HMGB1 might participate in pancreas and ileum injury in SAP. Growth hormone and somatostatin might play a therapeutic role in the inflammatory response of SAP.
Assuntos
Hormônio do Crescimento/metabolismo , Proteína HMGB1/metabolismo , Pâncreas/patologia , Pancreatite Necrosante Aguda/etiologia , Somatostatina/metabolismo , Animais , Edema/patologia , Endotoxinas/sangue , Expressão Gênica , Proteína HMGB1/genética , Hematoma/patologia , Íleo/lesões , Íleo/patologia , Interleucina-1beta/sangue , Interleucina-6/sangue , Masculino , Microscopia Eletrônica de Transmissão , Infiltração de Neutrófilos/fisiologia , Pâncreas/lesões , Pâncreas/metabolismo , Pancreatite Necrosante Aguda/metabolismo , Pancreatite Necrosante Aguda/patologia , RNA Mensageiro/isolamento & purificação , Distribuição Aleatória , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Taxa de Sobrevida , Fator de Necrose Tumoral alfa/sangueRESUMO
Challenged by the low salinity, 4 parts per thousand (4 ppt), for 72h, the survivals of swimming crabs (Portunus trituberculatus) were collected as the screened group (SG, tolerant to low salinity). Aiming at identifying the mechanism of low salinity tolerance, quantitative real-time PCR was employed to investigate the expression profiles of 4 HSP genes (HSP60, HSP70, HSP90-1, HSP90-2) in the hepatopancreas of wild (WG) and screened (SG) groups of P. trituberculatus exposed to low salinity (4 ppt). The results showed that 3 of the candidate genes (HSP60, HSP70, HSP90-1) exhibited similarly downregulated expression profiles in the first 3 h (P < 0.05), which became upregulated from 3 h to 72 h after being subjected to low salinity conditions. In contrast, the expression profile of the HSP90-2 gene was upregulated during the first 6 h for the WG, and during the first 12 h for the SG, after which it became downregulated. HSP90-1 and HSP90-2 were highly expressed at 12 h after low salinity challenge in the SG, but not the WG. The response of these 2 genes to salinity stress indicates their suitability as biomarkers to differentiate SG from WG crabs. The results indicate that HSP genes are involved in the adaptation of crabs to low salinity exposure, and that different HSPs have diverse functions in response to low salinity stress in P. trituberculatus. In addition, HSP expression in SG indicates that this group is more tolerant to low salinity conditions compared to WG.
Assuntos
Proteínas de Artrópodes/genética , Braquiúros/genética , Perfilação da Expressão Gênica , Proteínas de Choque Térmico/genética , Hepatopâncreas/metabolismo , Animais , Isoformas de Proteínas/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Salinidade , Tolerância ao Sal/genética , Natação , Fatores de TempoRESUMO
Accumulating evidence has indicated the importance of cancer stem cells in carcinogenesis. The goal of the present study was to determine the effect of low-dose cisplatin on enriched liver cancer stem cells (LCSCs). Human hepatoblastoma HepG2 cells were treated with concentrations of cisplatin ranging from 1 to 5 μg/mL. Cell survival and proliferation were evaluated using a tetrazolium dye (MTT) assay. LCSCs were identified using specific markers, namely aldehyde dehydrogenase-1 (ALDH1) and CD133. The percentage of ALDH1+ or CD133+ cells was examined by flow cytometric analysis. The expression of ALDH1 and/or CD133 in HepG2 cells was determined by immunocytochemical analysis. Low-dose cisplatin treatment significantly decreased cell survival in HepG2 cells after 24 or 72 h. However, the percentage of LCSCs in the surviving cells was greatly increased. The percentage of ALDH1+ or CD133+ cells was increased in a time- and dose-dependent manner after treatment with 1-4 μg/mL cisplatin, whereas 5 μg/mL cisplatin exposure slightly reduced the number of positive cells. These findings indicate that low-dose cisplatin treatment may efficiently enrich the LCSC population in HepG2 cells.
Assuntos
Humanos , Antineoplásicos/administração & dosagem , Proliferação de Células/efeitos dos fármacos , Cisplatino/administração & dosagem , Hepatoblastoma/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Células-Tronco Neoplásicas/efeitos dos fármacos , Antígenos CD/análise , Linhagem Celular Tumoral , Carcinogênese/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/uso terapêutico , Citometria de Fluxo , Glicoproteínas/análise , Hepatoblastoma/patologia , Imuno-Histoquímica , Isoenzimas/análise , Neoplasias Hepáticas/patologia , Células-Tronco Neoplásicas/citologia , Peptídeos/análise , Retinal Desidrogenase/análise , Sais de Tetrazólio , Biomarcadores Tumorais/análiseRESUMO
Accumulating evidence has indicated the importance of cancer stem cells in carcinogenesis. The goal of the present study was to determine the effect of low-dose cisplatin on enriched liver cancer stem cells (LCSCs). Human hepatoblastoma HepG2 cells were treated with concentrations of cisplatin ranging from 1 to 5 µg/mL. Cell survival and proliferation were evaluated using a tetrazolium dye (MTT) assay. LCSCs were identified using specific markers, namely aldehyde dehydrogenase-1 (ALDH1) and CD133. The percentage of ALDH1+ or CD133+ cells was examined by flow cytometric analysis. The expression of ALDH1 and/or CD133 in HepG2 cells was determined by immunocytochemical analysis. Low-dose cisplatin treatment significantly decreased cell survival in HepG2 cells after 24 or 72 h. However, the percentage of LCSCs in the surviving cells was greatly increased. The percentage of ALDH1+ or CD133+ cells was increased in a time- and dose-dependent manner after treatment with 1-4 µg/mL cisplatin, whereas 5 µg/mL cisplatin exposure slightly reduced the number of positive cells. These findings indicate that low-dose cisplatin treatment may efficiently enrich the LCSC population in HepG2 cells.
Assuntos
Antineoplásicos/administração & dosagem , Proliferação de Células/efeitos dos fármacos , Cisplatino/administração & dosagem , Hepatoblastoma/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Células-Tronco Neoplásicas/efeitos dos fármacos , Antígeno AC133 , Família Aldeído Desidrogenase 1 , Antígenos CD/análise , Biomarcadores Tumorais/análise , Carcinogênese/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/uso terapêutico , Citometria de Fluxo , Glicoproteínas/análise , Células Hep G2 , Hepatoblastoma/patologia , Humanos , Imuno-Histoquímica , Isoenzimas/análise , Neoplasias Hepáticas/patologia , Células-Tronco Neoplásicas/citologia , Peptídeos/análise , Retinal Desidrogenase/análise , Sais de TetrazólioRESUMO
The insulin-like growth factor-binding protein-5 (IGFBP-5) is one of the 6 members of the IGFBP family and is involved in the regulation of cell growth, apoptosis, and other IGF-stimulated signaling pathways. To determine the significance of IGFBP-5 in the Inner Mongolia Cashmere goat (Capra hircus), a hair follicle-specific expression vector of IGFBP-5, pCDsRed2-K-IGFBP5 (6.7 kb), was constructed by cloning IGFBP-5 downstream of the keratin-association protein (KAP)6-1 promoter and inserting this fragment into pCDsRed2, which contains a red fluorescent protein (DsRed) expression unit. Inner Mongolia Cashmere goat fetal fibroblast (GFb) cells were transfected with the expression vector by using Lipofectamine(TM) 2000. Cell clones that stably expressed red fluorescence were obtained after selection with Geneticin (G418). The transgene in the cell clones was examined by polymerase chain reaction to verify that exogenous DNA (pKAP6-1 and IGFBP-5) had integrated stably into GFb cells. These data suggest that this method can be used for the construction of a hair follicle-specific expression vector for functional genetic analyses and for obtaining stable transfection donor cells for nuclear transfer.
Assuntos
Cabras/genética , Folículo Piloso/metabolismo , Proteína 5 de Ligação a Fator de Crescimento Semelhante à Insulina/biossíntese , Transfecção , Animais , Animais Geneticamente Modificados , Embrião de Mamíferos/metabolismo , Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Cabras/metabolismo , Proteína 5 de Ligação a Fator de Crescimento Semelhante à Insulina/genéticaRESUMO
Methylation of the septin 9 gene (SEPT9) occurs in higher frequency in colorectal cancer (CRC) compared to control samples, which suggests that SEPT9 methylation is a useful biomarker for screening CRC. However, the methylation status of SEPT9 in Chinese CRC patients is scarcely reported. In the present study, SEPT9 methylation was tested in CRC tissues obtained from a Chinese population and correlations with pathological characteristics were investigated. The methylation status of SEPT9 was detected using methylation-specific polymerase chain reaction (PCR)-denaturing high-performance liquid chromatography (MSP-DHPLC) in 234 colorectal tissues (172 cases, 62 controls). Samples were sequenced to confirm the results from MSP-DHPLC. The chi-squared test was used to analyze the correlation of SEPT9 gene methylation status and pathological characteristics in CRCs. SEPT9 gene methylation was detected in 152 of 172 (88.4%) cases of verified CRC and in 4 of 62 (6.5%) healthy controls (χ(2) = 137.62, P < 0.001). There was no association between the methylation status of SEPT9 and age, gender, Duke's stage, TNM stage, differentiation, and site of cancer (P > 0.05). Our results suggest that SEPT9 gene methylation is a valuable biomarker for screening CRC in the Chinese population.
Assuntos
Neoplasias Colorretais/genética , Metilação de DNA/genética , Detecção Precoce de Câncer , Septinas/genética , Idoso , Povo Asiático , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estadiamento de NeoplasiasRESUMO
Complementary DNA (cDNA) is valuable for investigating protein structure and function in the study of life science, but it is difficult to obtain by traditional reverse transcription. We employed a novel strategy to clone human leukemia inhibitory factor (hLIF) gene cDNA from genomic DNA, which was directly isolated from the mucous membrane of mouth. The hLIF sequence, which is 609 bp long and is composed of three exons, can be acquired within a few hours by amplifying each exon and splicing all of them using overlap-PCR. This new approach developed is simple, time- and cost-effective, without RNA preparation or cDNA synthesis, and is not limited to the specific tissues for a particular gene and the expression level of the gene.