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1.
Adv Exp Med Biol ; 1169: 225-242, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31487027

RESUMO

Germ cells transfer genetic materials from one generation to the next, which ensures the continuation of the species. Spermatogenesis, the process of male germ cell production, is one of the most productive systems in adult tissues. This high productivity depends on the well-coordinated differentiation cascade in spermatogonia, occurring via their synchronized cell division and proliferation. Spermatogonial stem cells (SSCs) are responsible for maintaining the spermatogonial population via self-renewal and the continuous generation of committed progenitor cells that differentiate into spermatozoa. Like other stem cells in the body, SSCs are defined by their self-renewal and differentiation abilities. A functional transplantation assay, in which these biological properties of SSCs can be quantitatively evaluated, was developed using mice, and the cell surface characteristics and intracellular marker gene expression of murine SSCs were successfully determined. Another approach to elucidate SSC identity is a cell lineage-tracing experiment using transgenic mice, which can track the SSC behavior in the testes. Recent studies using both these experimental approaches have revealed that the SSC identity changed depending upon the developmental, homeostatic, and regenerative circumstances. In addition, single-cell transcriptomic analyses have further indicated the instability of marker gene expression in SSCs. More studies are needed to unify the results of the determination of SSC identity based on the functional properties and accumulating transcriptomic data of SSCs, to elucidate the functional interaction between SSC behavior and gene products and illustrate the conserved features of SSCs amidst their heterogeneity. Furthermore, the deterministic roles of distinct SSC niches under different physiological conditions in the SSC heterogeneity and its causal regulators must also be clarified in future studies.


Assuntos
Células-Tronco Germinativas Adultas , Espermatogônias , Animais , Células Cultivadas , Masculino , Camundongos , Espermatogênese , Espermatogônias/citologia , Testículo/citologia
2.
Adv Exp Med Biol ; 1169: 243-256, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31487028

RESUMO

Heterogeneity among different subpopulations of human umbilical cord mesenchymal stem cell (hUCMSCs) lines is an ubiquitous phenomenon, with such variability being related to several factors including the identity of the individual donor, tissue source (Wharton's jelly vs. umbilical cord blood), culture conditions, as well as random variations in the cloning expansion process. In this chapter, we provide a general overview on the sources as well as available experimental techniques for proper identification of heterogeneity in hUCMSCs. Finally, we provide a brief discussion on the current scientific evidence regarding the potential superiority of subpopulations of hUCMSCs for specific clinical applications. Taking into account the exponential growth on the available experimental data on hUCMSCs in the past few years, this chapter is not intended to be comprehensive in nature, but rather is intended to provide a general overview about the central role which the topic of heterogeneity has in both basic science and clinical research in umbilical cord stem cells.


Assuntos
Células-Tronco Mesenquimais , Cordão Umbilical , Diferenciação Celular , Células Cultivadas , Sangue Fetal/citologia , Humanos , Células-Tronco Mesenquimais/citologia , Cordão Umbilical/citologia , Geleia de Wharton
3.
Acta Virol ; 63(3): 270-277, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31507192

RESUMO

Orf, also called contagious ecthyma or contagious pustular dermatitis, is a significant zoonotic disease that primarily affects goat and sheep globally. Currently, the infection by orf virus (ORFV) has been observed in different host species worldwide, including China. Here, a suspected outbreak of orf infection in a goat farm in Anhui Province in 2018 was investigated. Through PCR, electron microscopy, and cell culture techniques, we confirmed that the outbreak was caused by ORFV. Consequently, the orf virus strain was named the AH/LA/2018 strain. The amplified and sequenced ORFV011 (B2L) and ORFV059 (F1L) genes were used to construct phylogenetic trees to elucidate the genetic characteristics of the ORFV and the molecular epidemiology of orf. The present study is the first systematic evolution analysis of the ORFV strain isolated in Anhui Province. The results of this study will be helpful to better understand the characteristics of ORFV, to help prevent and control the transmission of ORFV at an early stage in China. Keywords: Anhui Province; goat; orf virus; phylogenetic analysis.


Assuntos
Ectima Contagioso , Vírus do Orf , Filogenia , Animais , Células Cultivadas , China/epidemiologia , Ectima Contagioso/virologia , Genes Virais/genética , Doenças das Cabras/epidemiologia , Doenças das Cabras/virologia , Cabras , Microscopia Eletrônica de Transmissão , Vírus do Orf/classificação , Vírus do Orf/ultraestrutura , Reação em Cadeia da Polimerase , Ovinos , Doenças dos Ovinos/epidemiologia , Doenças dos Ovinos/virologia
5.
Rev Assoc Med Bras (1992) ; 65(8): 1067-1073, 2019 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-31531603

RESUMO

OBJECTIVE: Diabetes is a risk factor for acute kidney injury (AKI). However, its mechanism of pathogenesis has not been elucidated. The aim of the study was to investigate the role of inflammation and the toll-like receptor 7 (TLR7) in ischemic AKI for diabetes. METHODS: A high glucose hypoxia-reoxygenation model of human renal tubular epithelial (HK-2) cells was used to generate AKI induced by ischemia-reperfusion in diabetes. The activity of cells was measured by CCK-8 assay and LDH activity. Inflammatory cytokines were assessed by ELISA. TLR7, MyD88, and NF-κB expressions were examined by western blotting. Apoptosis was evaluated by flow cytometry. RESULTS: The high glucose group and low glucose group were subjected to hypoxia-reoxygenation. The low glucose group developed only mild cell damage, apoptosis, and inflammatory response. In contrast, an equivalent hypoxia-reoxygenation injury provoked severe cell damage, apoptosis, and inflammatory response in the high glucose group. Expression of TLR7 and its related proteins were measured in the high glucose group before and after hypoxia-reoxygenation. The high glucose group exhibited more significant increases in TLR7 expression following hypoxia-reoxygenation than the low glucose group. In addition, the expression of TLR7 and its related proteins after hypoxia-reoxygenation were higher in the high glucose group than in the low glucose group. Inhibition of TLR7 provides significant protection against ischemic injury in diabetes. CONCLUSION: Our results suggest that diabetes increases the vulnerability to ischemia-induced renal injury. This increased vulnerability originates from a heightened inflammatory response involving the TLR7 signal transduction pathway.


Assuntos
Lesão Renal Aguda/metabolismo , Diabetes Mellitus/metabolismo , Isquemia/metabolismo , Receptor 7 Toll-Like/metabolismo , Lesão Renal Aguda/fisiopatologia , Células Cultivadas , Diabetes Mellitus/fisiopatologia , Citometria de Fluxo , Humanos , Isquemia/fisiopatologia , RNA Interferente Pequeno , Transdução de Sinais , Receptor 7 Toll-Like/fisiologia , Transfecção
6.
Rev Assoc Med Bras (1992) ; 65(8): 1122-1127, 2019 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-31531613

RESUMO

Melatonin is known for its effects on both the sleep and reproductive system of mammals. The latter has melatonin receptors type 1 and 2, which act to regulate, among other things, cyclic AMP. Notwithstanding all the literature data, there is still no sound knowledge or a clear understanding of the hormone's action on the physiology of ovarian follicular cells. OBJECTIVE To review and evaluate studies about melatonin action on the ovarian granulosa/theca interna cells from the literature. METHODS The systematic review was carried out according to the PRISMA recommendations. The MEDLINE and Cochrane primary databases were consulted with the use of specific terms. There was no limitation on language or publication year. RESULTS Seven papers about melatonin action on granulosa cells were selected. The following can be attributed to the hormone's effects: a) progesterone increase in culture medium; b) increased estrogen production; c) antagonistic action on estrogen; d) improvement in cell quality resulting in improved embryo and higher pregnancy rates; e) improved cell proliferation via MAPK; f) reduction of free radicals. Nevertheless, there are contrarian papers reporting a reduction in progesterone production. Melatonin interferes in sex steroid production, boosting progesterone output. Such action may help improve oocyte quality.


Assuntos
Melatonina/farmacologia , Oócitos/efeitos dos fármacos , Folículo Ovariano/efeitos dos fármacos , Células Cultivadas , Feminino , Células da Granulosa/efeitos dos fármacos , Humanos , Oócitos/crescimento & desenvolvimento , Gravidez , Progesterona/antagonistas & inibidores , Células Tecais/efeitos dos fármacos
7.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 35(7): 583-588, 2019 Jul.
Artigo em Chinês | MEDLINE | ID: mdl-31537241

RESUMO

Objective To explore the effect of prostaglandin E2 (PGE2) on the phenotype and chemotaxis of mouse bone marrow-derived dendritic cells (BMDCs). Methods BMDCs isolated from murine bone marrow and cultured in vitro were divided into 6 groups (0 µg/mL PGE2 group, 1 µg/mL PGE2 group, 5 µg/mL PGE2 group, 0 µg/mL PGE2 plus LPS group, 1 µg/mL PGE2 plus LPS group, 5 µg/mL PGE2 plus LPS group). The expression of surface makers CD40, CD86, major histocompatibility complex II (MHCII), CC chemokine receptor 7 (CCR7) were detected by flow cytometry. The expression of CCR7 protein was detected by Western blot analysis. The migration ability of BMDCs was detected by TranswellTM assay. The survival rate of BMDCs was detected by CCK-8 assay. Results PGE2 of 1 µg/mL increased the expression of surface molecules on BMDCs and promoted the migration ability of BMDCs. PGE2 of 5 µg/mL reduced the expression of surface molecules on BMDCs and inhibited the migration ability of BMDCs. The change of PGE2 concentration did not affect the survival rate of BMDCs. Conclusion PGE2 was demonstrated a dual regulatory effect on the migration of BMDCs. Low concentration of PGE2 can promote the migration ability of BMDCs, while high concentration of PGE2 shows contrary effect.


Assuntos
Medula Óssea , Movimento Celular , Células Dendríticas/citologia , Dinoprostona/farmacologia , Animais , Antígeno B7-2/metabolismo , Antígenos CD40/metabolismo , Células Cultivadas , Células Dendríticas/efeitos dos fármacos , Antígenos de Histocompatibilidade Classe II/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Receptores CCR7/metabolismo
8.
Zhongguo Yi Xue Ke Xue Yuan Xue Bao ; 41(4): 443-451, 2019 Aug 30.
Artigo em Chinês | MEDLINE | ID: mdl-31484604

RESUMO

Objective To analyze the differences in biological functions between bone marrow(BM)-derived CD106 +mesenchymal stem cells(MSCs)and the CD106 - subgroup. Methods The MSCs from normal BM were isolated and expanded.The subgroups of CD106 + and CD106 -MSCs were sorted.The cell proliferation and adhesion functions,chemotactic activities,adipogenic and osteogenic potentials,senescence,and senescence protein 21(p21)were detected.The capacity of translocation into nucleus of nuclear factor-kappa B(NF-κB)when stimulated by tumor necrosis factor(TNF-α)was measured. Results The proliferative ability was higher in CD106 +MSCs than that in CD106 -MSCs.In 48 hours,the value of optical density(OD)was significantly higher in CD106 +MSCs than that in CD106 - subgroup(1.004±0.028 vs. 0.659±0.023,t=3.946,P=0.0225).In 72 hours,this phenomenon was even more pronounced(2.574±0.089 vs. 1.590±0.074,t=11.240,P=0.0000).The adhesive capacity of CD106 +MSCs was significantly stronger than that of CD106 - subgroup(0.648±0.018 vs. 0.418±0.023,t=7.869,P=0.0002).Besides,the metastasis ability of CD106 +MSCs were significantly stronger than that of CD106 - subgroup(114.500±4.481 vs.71.000±4.435,t=6.900,P=0.0005).The CD106 +MSCs had signifcnatly lower proportions of senescent cells.The expression of aging protein p21 in CD106 +MSCs was significantly lower than that in CD106 -MSCs [(17.560±1.421)% vs.(45.800±2.569)%,t=9.618,P=0.0000].Furthermore,there were no visible pigmenting cells after ß-galactosidase staining in CD106 +MSCs subgroup.However,in CD106 -MSCs,some colored green cells were detected.The rate of NF-κB translocation into nucleus after stimulated by TNF-α was significantly higher in CD106 +MSCs than CD106 - MSCs [(37.780±3.268)% vs.(7.30±1.25)%,t=8.713,P=0.0001]. Conclusion Bone marrow-derived CD106 +MSCs possess more powerful biological functions than CD106 -MSCs.


Assuntos
Células da Medula Óssea/citologia , Células-Tronco Mesenquimais/citologia , Molécula 1 de Adesão de Célula Vascular/metabolismo , Adesão Celular , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Humanos , NF-kappa B/metabolismo , Transporte Proteico , Fator de Necrose Tumoral alfa/farmacologia
9.
Zhongguo Yi Xue Ke Xue Yuan Xue Bao ; 41(4): 524-528, 2019 Aug 30.
Artigo em Chinês | MEDLINE | ID: mdl-31484616

RESUMO

To compare the biological functions of astrocytes cultured in vitro by two methods. Methods The primary astrocytes were cultured from rodent neonatal brain,whereas the differentiated astrocytes were prepared by differentiating neural stem cells with fetal bovine serum.The morphologies of these two different types of astrocytes were observed under microscope and the expression of glial fibrillary acidic protein(GFAP),an astrocyte-specific marker,was detected by immunofluorescence staining after treatment with 10 cytokines.Changes in GFAP,glutamate synthetase(GS),glutamate-aspartic acid transporter(xCT),neuregulin-1(NRG),N-methyl-D-aspartic acid receptor(NMDA),lipoprotein lipase(LPL)were detected and compared. Results The morphologies and GFAP expression differed between these two astrocyte types.Microarray showed that the expressions of GFAP,GS,xCT,NRG,NMDA,and LPL were significantly higher in primary astrocytes than in differentiated astrocytes.None of these 10 cytokines increased the expression of GFAP in primary astrocytes,whereas treatment with transforming growth factor-ß(TGF-ß)significantly increased the expression of GFAP in the differentiated astrocytes. Conclusion Compared with the differentiated astrocytes,the primary astrocytes are more similar to reactive astrocytes,and TGF-ß can promote the transition of differentiated cells to reactive cells.


Assuntos
Astrócitos/citologia , Diferenciação Celular , Animais , Animais Recém-Nascidos , Células Cultivadas , Proteína Glial Fibrilar Ácida/metabolismo , Células-Tronco Neurais/citologia , Roedores , Fator de Crescimento Transformador beta/farmacologia
10.
Braz Oral Res ; 33: e042, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31508725

RESUMO

This study evaluated the cytotoxicity and biocompatibility of a new bioceramic endodontic sealer (i.e., Sealer Plus BC) in comparison with those of MTA Fillapex and AH Plus. L929 fibroblasts were cultured and Alamar Blue was used to evaluate cell viability of diluted extracts (1:50, 1:100, and 1:200) from each sealer at 24 h. Polyethylene tubes that were filled with material or empty (as a control) were implanted in the subcutaneous tissue of rats. The rats were killed after 7 and 30 d (n = 8), and the tubes were removed for histological analysis. Parametric data was analyzed using a one-way ANOVA test, and nonparametric data was analyzed via the Kruskal-Wallis test followed by the Dunn test (p < 0.05). A reduction in cell viability was observed in the extracts that were more diluted for Sealer Plus BC when compared to that of Control and AH Plus (p < 0.05). However, the 1:50 dilution of the Sealer Plus BC was similar to that of the Control (p > 0.05). Conversely, more diluted extracts of MTA Fillapex (1:200) and AH Plus (1:100 and 1:200) were similar to the Control (p > 0.05). Histological analysis performed at 7 d did not indicate any significant difference between tissue response for all materials, and the fibrous capsule was thick (p > 0.05). At 30 d, Sealer Plus BC was similar to the Control (p > 0.05) and MTA Fillapex and AH Plus exhibited greater inflammation than the Control (p < 0.05). The fibrous capsule was thin for the Control and for most specimens of Sealer Plus BC and AH Plus. Thus, Sealer Plus BC is biocompatible when compared to MTA Fillapex and AH Plus, and it is less cytotoxic when less-diluted extracts are used.


Assuntos
Cimentos para Ossos/química , Hidróxido de Cálcio/química , Cerâmica/química , Materiais Restauradores do Canal Radicular/química , Compostos de Alumínio/química , Animais , Materiais Biocompatíveis , Cimentos para Ossos/farmacologia , Cimentos para Ossos/toxicidade , Compostos de Cálcio/química , Hidróxido de Cálcio/farmacologia , Hidróxido de Cálcio/toxicidade , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas/efeitos dos fármacos , Combinação de Medicamentos , Resinas Epóxi/química , Fibroblastos/efeitos dos fármacos , Técnicas In Vitro , Inflamação , Masculino , Teste de Materiais , Óxidos/química , Ratos Wistar , Materiais Restauradores do Canal Radicular/toxicidade , Silicatos/química , Tela Subcutânea/patologia
11.
Bratisl Lek Listy ; 120(9): 686-689, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31475555

RESUMO

BACKGROUND: The lipografting is increasingly used in the field of plastic surgery. Widely used harvesting technique of fatderived stem-cells is lipoaspiration. There exist two big streams of fat harvesting for lipografting: mechanical liposuction and manual liposuction. METHODS: Two harvested specimens were compared in this prospective blind study in the means of stem-cells viability and their ability to grow in cell-cultures. Techniques to compare were: manual lipoaspiration with 50 ml syringe and WAL (water-jet assisted liposuction). RESULTS: Twenty specimens from ten patients were investigated in the tissue bank. There were no differences in the amount of live stem-cells between two groups. Also no differences were found between both harvesting techniques in the mean of cell ability to grow in cell-cultures. CONCLUSION: It can be concluded that there are no statistically significant differences in the number, vitality and viability of stem cells when comparing two ways of mesenchymal stem cell collection, both manual and machine sampling (WAL). When cultured in vitro, both samples collected from each patient also appeared to be able to multiply with no statistical differences (Tab. 2, Fig. 2, Ref. 18).


Assuntos
Tecido Adiposo/citologia , Lipectomia/métodos , Células-Tronco Mesenquimais/citologia , Células Cultivadas , Humanos , Estudos Prospectivos
12.
Zhonghua Yi Xue Za Zhi ; 99(35): 2745-2749, 2019 Sep 17.
Artigo em Chinês | MEDLINE | ID: mdl-31550796

RESUMO

Objective: To investigatea cellular/molecular mechanism of the CD40/TRAF1 signalling pathway involved in Rheumatoid arthritis (RA). Methods: 16 patients with active RA and 9 patients with Fractures who underwent total knee or hip replacement in The First Affiliated Hospital of Soochow University were included in the study. Synovial tissues (ST) and serum were obtained from each patient. The CD40, TRAF1, NF-κB p65 were detected by ELISA and Immunohistochemistry in serum and tissue respectively. Real time-PCR (RT-PCR) was applied to measure NF-κB-related gene expression. Results: CD40 and TRAF1 positive area (%) in RA patients were 28.7±5.4, 34.3±4.8 respectively, which were significantly higher (P<0.05) than Fracture controls (21.2±9.5, 21.6±8.7 respectively). The expression of total NF-κB p65, and phospho-NF-κB p65 proteins, as well as NF-κB-related gene expression, including cytokines (TNFα, IL-6), chemokines (MCP-1),and adhesion molecules (ICAM-1) were significantly higher in the ST of RA patients compared to Fracture controls. Conclusion: It is thus possible that the CD40/TRAF1 pathway acted as a positive regulator through NF-κB activation and NF-κB-dependent proinflammatory genes in RA.


Assuntos
Artrite Reumatoide/genética , Antígenos CD40/metabolismo , Transdução de Sinais , Fator 1 Associado a Receptor de TNF/metabolismo , Fator de Transcrição RelA/metabolismo , Antígenos CD40/genética , Células Cultivadas , Expressão Gênica , Humanos , Membrana Sinovial , Fator 1 Associado a Receptor de TNF/genética , Fator de Transcrição RelA/genética
13.
Nihon Yakurigaku Zasshi ; 154(3): 133-137, 2019.
Artigo em Japonês | MEDLINE | ID: mdl-31527363

RESUMO

Hydrogen sulfide (H2S) has been focused as a biological mediator, which modulates signal transduction and protects cells and tissues from oxidative stress. H2S is also expected as a neuroprotectant because it has a neuroprotective activity. Endogenous H2S is mainly generated from L-cysteine. However, it is difficult to use L-cysteine as a neuroprotectant because of its neurotoxicity. In 2013, a novel biogenesis pathway of H2S from D-cysteine has been identified. In this pathway, D-amino acid oxidase (DAO) converts D-cysteine to 3-mercaptopyruvate (3MP), followed by the generation of H2S from 3MP by 3-mercaptopyrvate sulfurtransferase. DAO is especially abundant in cerebellum among various brain regions and mediates efficient generation of H2S from D-cysteine in the cerebellar tissues. In addition, D-cysteine has more potent neuroprotective activity in cerebellar primary neurons than L-cysteine. Cerebella Purkinje cells (PCs) are characterized by the highly-branched dendrites and are important for cerebellar functions. The dendritic shrinkage and degeneration of PCs are frequently observed in patients and model mice of cerebellar ataxias. We revealed that D-cysteine enhanced dendritic development of primary cultured PCs, but L-cysteine impaired the dendritic development. This effect of D-cysteine was inhibited by DAO inhibitors and reproduced by 3MP and a H2S donor, suggesting that this enhancement of dendritic development is caused by the production of H2S from D-cysteine. Taken together, D-cysteine would be available as a neuroprotectant against cerebellar ataxias, which are accompanied with dendritic shrinkage of cerebellar PCs.


Assuntos
Cisteína/metabolismo , Sulfeto de Hidrogênio/metabolismo , Neurônios/citologia , Fármacos Neuroprotetores/metabolismo , Animais , Células Cultivadas , D-Aminoácido Oxidase/antagonistas & inibidores , D-Aminoácido Oxidase/metabolismo , Humanos , Camundongos , Neurogênese , Estresse Oxidativo , Células de Purkinje/citologia
14.
Gene ; 720: 144096, 2019 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-31476405

RESUMO

Biologically active materials and polymeric materials used in tissue engineering have been one of the most attractive research areas in the past decades, especially the use of easily accessible materials from the patients that reduces or eliminates any patient's immune response. In this study, electrospun nanofibrous scaffolds were fabricated by using polyvinyl-alcohol (PVA), chitosan and hydroxyapatite (HA) polymers and platelet-rich plasma (PRP) as a bioactive substance isolated from human blood. Fabricated scaffold's structure and cytotoxicity were evaluated using scanning electron microscope and MTT assays. Scaffolds osteoinductivity was investigated by osteogenic differentiation of the mesenchymal stem cells (MSCs) at the in vitro level and then its osteoconductivity was examined by implanting at the critical-sized rat calvarial defect. The in vitro results showed that scaffolds have a good structure and good biocompatibility. Alkaline phosphatase activity, calcium content and gene expression assays were also demonstrated that their highest amount was detected in MSCs-seeded PVA-chitosan-HA(PRP) scaffold. For this reason, this scaffold alone and along with the MSCs was implanted to the animal defects. The in vivo results demonstrated that in the animals implanted with PVA-chitosan-HA(PRP), the defect was repaired to a good extent, but in those animals that received MSCs-seeded PVA-chitosan-HA(PRP), the defects was almost filled. It can be concluded that, PVA-chitosan-HA(PRP) alone or when stem cells cultured on them, has a great potential to use as an effective bone implant.


Assuntos
Diferenciação Celular , Nanofibras/química , Osteogênese , Plasma Rico em Plaquetas/química , Procedimentos Cirúrgicos Reconstrutivos , Crânio/cirurgia , Animais , Células Cultivadas , Quitosana/química , Durapatita/química , Masculino , Células-Tronco Mesenquimais/citologia , Álcool de Polivinil/química , Ratos , Ratos Sprague-Dawley , Engenharia Tecidual , Tecidos Suporte
15.
Chem Biol Interact ; 312: 108817, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31499053

RESUMO

Aconitine might have reproductive toxicity and the effects of aconitine on androgen synthesis in Leydig cells remain unclear. Here, we explore how aconitine affects androgen synthesis and metabolism in rat immature Leydig cells in vitro. Immature Leydig cells were isolated from 35-day-old male Sprague Dawley rats and cultured with 0-50 µM aconitine for 3 h in combination with LH, 8Br-cAMP, 22R-hydroxycholesterol, pregnenolone, progesterone, androstenedione, testosterone, and dihydrotestosterone, respectively. Medium androgens were measured. The levels of Leydig cell mRNAs, Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, Srd5a1, and Akr1c14, were measured by qPCR. ROS and apoptosis were determined after 24-h aconitine treatment. Aconitine inhibited basal androgen production in Leydig cells at 0.05 µM and the higher concentrations. Aconitine blocked pregnenolone, progesterone, and androstenedione mediated androgen outputs without affecting 22R-hydroxycholesterol-mediated androgen production at 5 µM. Aconitine also inhibited LH and 8Br-cAMP stimulated androgen outputs at 5 µM. Further investigation showed that aconitine blocked androgen synthesis via down-regulating the expression of Scarb1, Hsd3b1, Cyp17a1, and Hsd17b3. At 50 µM, aconitine also induced ROS generation and increased apoptotic rate of Leydig cells. Aconitine lowered serum testosterone levels at 1.5 mg/kg after 7 days of oral exposure from postnatal day 35. In conclusion, aconitine inhibits androgen synthesis.


Assuntos
Aconitina/farmacologia , Androgênios/metabolismo , Regulação para Baixo/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Células Cultivadas , Células Intersticiais do Testículo/citologia , Células Intersticiais do Testículo/efeitos dos fármacos , Células Intersticiais do Testículo/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Receptores do LH/genética , Receptores do LH/metabolismo , Receptores Depuradores Classe B/genética , Receptores Depuradores Classe B/metabolismo , Testosterona/sangue , Testosterona/farmacologia
16.
Artigo em Chinês | MEDLINE | ID: mdl-31495106

RESUMO

Objective: To study the effect of particulate matter 2.5 (PM(2.5)) on oncogene expression in human bronchial epithelial (HBE) cells. Methods: HBE cells were selected as the study subjects, and PM(2.5) treatment group (10 µg/ml and 50 µg/ml) , negative control group and positive control group (10 µmol/L Cr(6+)) were set. CCK8 assay was used to test the IC(50) value of PM(2.5). HBE cells were treated with PM(2.5) for 24 h at 10 µg/ml and 50 µg/ml, additionally, cells were treated with blank as negative control, 10 µmol/L Cr(6+) as a positive control for 24 h. After the treatment, mRNA expression of oncogenes including c-myc, c-fos, k-ras and p53 were detected by fluorescent quantitative RT-PCR, the protein expression of oncogenes were detected with western blot. Results: The IC(50) value of PM(2.5) in HBE cells is 70.12 µg/ml. The qRT-PCR data showed that compared with the control group, the expression level of c-myc gene increased by respectively 500.1%、780.7%、305.3% after exposure to 10、50 µg/ml PM(2.5) and positive control group; c-fos gene increased respectively 34.0%、76.7%、131.3% after exposure to 10、50 µg/ml PM(2.5) and positive control group; k-ras gene increased respectively 50.3%、107.0%、49.7% after exposure to 10、50 µg/ml PM(2.5) and positive control group; p53 gene decreased by 28.3%、28.7%、59.7% after exposure to 10、50 µg/ml PM(2.5) and positive control group. The western blot results showed that compared with the control group, c-myc protein increased respectively 29.7%、77.3% after exposure to 50 µg/ml PM(2.5) and positive control group; c-fos protein increased respectively 200.3%、137.0% after exposure to 50 µg/ml PM(2.5) and positive control group; k-ras protein increased respectively 106.3%、130.3%、116.7% after exposure to 10、50 µg/ml PM(2.5) and positive control group; p53 protein decreased by 43.7%、53.3%、52.1% after exposure to 10、50 µg/ml PM(2.5) and positive control group. Conclusion: PM(2.5) could promote the expression of oncogenes in HBE cells, the carcinogenicity of haze might be related to promotion of oncogenes expression induced by PM(2.5).


Assuntos
Células Epiteliais/efeitos dos fármacos , Oncogenes , Material Particulado/toxicidade , Brônquios/citologia , Células Cultivadas , Humanos , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteína Supressora de Tumor p53/genética
17.
J Agric Food Chem ; 67(37): 10285-10295, 2019 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-31443611

RESUMO

Fluoride (F) is capable of promoting abnormal proliferation and differentiation in primary cultured mouse osteoblasts (OB cells), although the underlying mechanism responsible remains rare. This study aimed to explore the roles of wingless and INT-1 (Wnt) signaling pathways and screen appropriate doses of calcium (Ca2+) to alleviate the sodium fluoride (NaF)-induced OB cell toxicity. For this, we evaluated the effect of dickkopf-related protein 1 (DKK1) and Ca2+ on mRNA levels of wingless/integrated 3a (Wnt3a), low-density lipoprotein receptor-related protein 5 (LRP5), dishevelled 1 (Dv1), glycogen synthase kinase 3ß (GSK3ß), ß-catenin, lymphoid enhancer binding factor 1 (LEF1), and cellular myelocytomatosis oncogene (cMYC), as well as Ccnd1 (Cyclin D1) in OB cells challenged with 10-6 mol/L NaF for 24 h. The demonstrated data showed that F significantly increased the OB cell proliferation rate. Ectogenic 0.5 mg/L DKK1 significantly inhibited the proliferation of OB cells induced by F. The mRNA expression levels of Wnt3a, LRP5, Dv1, LEF1, ß-catenin, cMYC, and Ccnd1 were significantly increased in the F group, while significantly decreased in the 10-6 mol/L NaF + 0.5 mg/L DKK1 (FY) group. The mRNA expression levels of Wnt3a, LRP5, ß-catenin, and cMYC were significantly decreased in the 10-6 mol/L NaF + 2 mmol/L CaCl2 (F+CaII) group. The protein expression levels of Wnt3a, Cyclin D1, cMYC, and ß-catenin were significantly increased in the F group, whereas they were decreased in the F+CaII group. However, the mRNA and protein expression levels of GSK3ß were significantly decreased in the F group while significantly increased in the F+CaII group. In summary, F activated the canonical Wnt/ß-catenin pathway and changed the related gene expression and ß-catenin protein location in OB cells, promoting cell proliferation. Ca2+ supplementation (2 mmol/L) reversed the expression levels of genes and proteins related to the canonical Wnt/ß-catenin pathway.


Assuntos
Cálcio/metabolismo , Fluoretos/efeitos adversos , Osteoblastos/efeitos dos fármacos , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Suplementos Nutricionais/análise , Glicogênio Sintase Quinase 3 beta/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Masculino , Camundongos , Osteoblastos/classificação , Osteoblastos/metabolismo , Proteínas Wnt/genética , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/genética
18.
Zhonghua Xin Xue Guan Bing Za Zhi ; 47(7): 554-560, 2019 Jul 24.
Artigo em Chinês | MEDLINE | ID: mdl-31365997

RESUMO

Objective: To investigate the role of piperine on the transformation of endothelial cells into fibroblasts. Methods: Cultured human umbilical vein endothelial cells (HUVECs, 4-6 passage) were used for the main experiments. The transformation models of endothelial cells into fibroblasts were induced by transforming growth factor ß (TGF-ß) stimulation. HUVECs were divided into 6 groups: control group, TGF-ß group and 4 groups treated with various concentrations of piperine (1, 5, 10, 20 µmol/L). CKK-8 was used to detect cell proliferation. The CD31/α-smooth muscle actin (α-SMA) expression level was detected by fluorescent staining. The vascular endothelial cadherin (VE-cadherin)/vimentin expression was detected by immunofluorescence staining. RT-PCR was used detect the mRNA expressions of transformation markers. Western blot was used to detect the protein expression of snail and twist. Results: TGF-ß increased HUVECs proliferation (P<0.05), which could be significantly inhibited by 10 and 20 µmol/L of piperine, but not by 1 and 5 µmol/L of piperine. Immunofluorescence results demonstrated that TGF-ß increased HUVECs transformation to fibroblasts as shown by downregulated expression of endothelial markers CD31, VE-cadherin, and upregulated expression of α-SMA and vimentin, again, these effects could be attenuated by 10 and 20 µmol/L piperine. The expression levels of collagen type Ⅰ and type Ⅲ were significantly higher in TGF-ß group than in control group (P<0.05), significantly lower in TGF-ß+10 µmol/L piperine group and TGF-ß+20 µmol/L piperine group than in TGF-ß group (P<0.05).In addition, RT-PCR results showed that TGF-ß increased mRNA expression of transformation markers (snail1, snail2, twist1, twist2), while 10 and 20 µmol/L of piperine could significantly downregulated the mRNA expressions of these markers. The protein expression levels of snail and twist were significantly higher in TGF-ß group than in control group (both P<0.05), which was significantly lower in TGF-ß+20 µmol/L piperine group than in TGF-ß group (both P<0.05). Conclusions: Piperine can inhibit the transformation of endothelial cells into fibroblasts. This effect might be viewed as one of the potential mechanisms of reduced myocardial fibrosis post piperine treatment.


Assuntos
Fibroblastos , Actinas , Alcaloides , Benzodioxóis , Células Cultivadas , Células Endoteliais , Humanos , Piperidinas , Alcamidas Poli-Insaturadas , Fator de Crescimento Transformador beta1
19.
Zhonghua Xin Xue Guan Bing Za Zhi ; 47(7): 561-569, 2019 Jul 24.
Artigo em Chinês | MEDLINE | ID: mdl-31365998

RESUMO

Objective: To investigate the impact of homocysteine inducible endoplasmic reticulum(ER) protein with ubiquitin like domain 1 protein (Herpud1) in the homocysteine (Hcy) -induced phenotypic switching of vascular smooth muscle cells (VSMCs). Methods: VSMCs were derived from thoracic aortic artery of male Sprague Dawley rats and cultured VSMCs (4-7 passage) were treated with various concentrations of Hcy (0, 100, 500 and 1 000 µmol/L) and applied to immunofluorescence to observe the morphological changes of VSMCs via SM-actin staining. Western blot was used to detect the expression of VSMCs phenotypic markers, including Osteopontin, Calponin and smooth muscle myosin heavy chain (SM-MHC) and the expression of endoplasmic reticulum stress (ERS) related proteins, including C/EBP-homologous protein (CHOP), inositol-requiring kinase 1 (IRE-1) and glucose regulating protein 78 (GRP78) in the absence and presence of non-selective inhibitor of ERS, 4-phenylbutyric acid (4-PBA, 2 mg/ml). The Herpud1 mRNA and protein levels were determined in Hcy-stimulated VSMCs treated with 4-PBA or transfected with specific siRNA targeting Herpud1. Results: Compared with the control group, SM-actin staining results showed that the shape of VSMCs treated with different concentrations of Hcy for 24 hours changed from long fusiform into round form, arrangement of myofilament became irregular and the most significant alteration was found in the 500 µmol/L Hcy group. After intervention of 24 hours, various concentration of Hcy increased protein expression of Osteopontin, and reduced Calponin and SM-MHC protein expressions in VSMCs (all P<0.05). In addition, the results showed that Hcy increased the expression of CHOP, IRE-1 and GRP78 in a dose-dependent manner, which could be reversed by 4-PBA treatment (all P<0.05). However, 4-PBA inhibited Hcy induced upregulation of Osteopontin and downregulation of Calponin and SM-MHC, suggesting that ERS was involved in Hcy-induced phenotypic switching of VSMCs. Herpud1 protein was mostly expressed in the cytoplasm and was also expressed in the nucli, both in the control, Hcy and Hcy+4-PBA groups. Moreover, Hcy increased mRNA and protein levels of Herpud1 (P<0.05), whereas treatment with 4-PBA could significantly reduce Hcy-induced upregulation of Herpud1 (P<0.05). Furthermore, knockdown of Herpud1 abrogated the effects of Hcy on VSMCs phenotype markers. Conclusion: Herpud1 plays an important role in Hcy-induced phenotypic switching of VSMCs.


Assuntos
Músculo Liso Vascular , Animais , Células Cultivadas , Homocisteína , Masculino , Miócitos de Músculo Liso , Fenótipo , Ratos , Ratos Sprague-Dawley
20.
Braz J Med Biol Res ; 52(8): e8318, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31411247

RESUMO

Currently, there is great clinical need for suitable synthetic grafts that can be used in vascular diseases. Synthetic grafts have been successfully used in medium and large arteries, however, their use in small diameter vessels is limited and presents a high failure rate. In this context, the aim of this study was to develop tissue engineering scaffolds, using poly(trimethylene carbonate-co-L-lactide) (PTMCLLA), for application as small diameter vascular grafts. For this, copolymers with varying trimethylene carbonate/lactide ratios - 20/80, 30/70, and 40/60 - were submitted to electrospinning and the resulting scaffolds were evaluated in terms of their physicochemical and biological properties. The scaffolds produced with PTMCLLA 20/80, 30/70, and 40/60 showed smooth fibers with an average diameter of 771±273, 606±242, and 697±232 nm, respectively. When the degradation ratio was evaluated, the three scaffold groups had a similar molecular weight (Mw) on the final day of analysis. PTMCLLA 30/70 and 40/60 scaffolds exhibited greater flexibility than the PTMCLLA 20/80. However, the PTMCLLA 40/60 scaffolds showed a large wrinkling and their biological properties were not evaluated. The PTMCLLA 30/70 scaffolds supported the adhesion and growth of mesenchymal stem cells (MSCs), endothelial progenitor cells, and smooth muscle cells (SMCs). In addition, they provided a spreading of MSCs and SMCs. Given the results, the electrospun scaffolds produced with PTMCLLA 30/70 copolymer can be considered promising candidates for future applications in vascular tissue engineering.


Assuntos
Prótese Vascular , Dioxanos/química , Poliésteres/química , Tecidos Suporte/química , Proliferação de Células , Células Cultivadas/citologia , Células Progenitoras Endoteliais/citologia , Humanos , Teste de Materiais , Células-Tronco Mesenquimais/citologia , Miócitos de Músculo Liso/citologia
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