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1.
Gut ; 69(1): 146-157, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-30723104

RESUMO

OBJECTIVE: We explored the hypothesis that TGR5, the bile acid (BA) G-protein-coupled receptor highly expressed in biliary epithelial cells, protects the liver against BA overload through the regulation of biliary epithelium permeability. DESIGN: Experiments were performed under basal and TGR5 agonist treatment. In vitro transepithelial electric resistance (TER) and FITC-dextran diffusion were measured in different cell lines. In vivo FITC-dextran was injected in the gallbladder (GB) lumen and traced in plasma. Tight junction proteins and TGR5-induced signalling were investigated in vitro and in vivo (wild-type [WT] and TGR5-KO livers and GB). WT and TGR5-KO mice were submitted to bile duct ligation or alpha-naphtylisothiocyanate intoxication under vehicle or TGR5 agonist treatment, and liver injury was studied. RESULTS: In vitro TGR5 stimulation increased TER and reduced paracellular permeability for dextran. In vivo dextran diffusion after GB injection was increased in TGR5-knock-out (KO) as compared with WT mice and decreased on TGR5 stimulation. In TGR5-KO bile ducts and GB, junctional adhesion molecule A (JAM-A) was hypophosphorylated and selectively downregulated among TJP analysed. TGR5 stimulation induced JAM-A phosphorylation and stabilisation both in vitro and in vivo, associated with protein kinase C-ζ activation. TGR5 agonist-induced TER increase as well as JAM-A protein stabilisation was dependent on JAM-A Ser285 phosphorylation. TGR5 agonist-treated mice were protected from cholestasis-induced liver injury, and this protection was significantly impaired in JAM-A-KO mice. CONCLUSION: The BA receptor TGR5 regulates biliary epithelial barrier function in vitro and in vivo through an impact on JAM-A expression and phosphorylation, thereby protecting liver parenchyma against bile leakage.


Assuntos
Sistema Biliar/fisiopatologia , Colestase Intra-Hepática/prevenção & controle , Receptores Acoplados a Proteínas-G/fisiologia , Animais , Bile/metabolismo , Ácidos e Sais Biliares/metabolismo , Moléculas de Adesão Celular/metabolismo , Células Cultivadas , Colestase Intra-Hepática/metabolismo , Impedância Elétrica , Epitélio/fisiopatologia , Ácidos Isonipecóticos/farmacologia , Ácidos Isonipecóticos/uso terapêutico , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oximas/farmacologia , Oximas/uso terapêutico , Permeabilidade , Fosforilação/fisiologia , Receptores de Superfície Celular/metabolismo , Receptores Acoplados a Proteínas-G/agonistas , Transdução de Sinais/fisiologia , Proteínas de Junções Íntimas/metabolismo
2.
Gut ; 69(1): 133-145, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31409605

RESUMO

OBJECTIVE: The Fragile X mental retardation (FMR) syndrome is a frequently inherited intellectual disability caused by decreased or absent expression of the FMR protein (FMRP). Lack of FMRP is associated with neuronal degradation and cognitive dysfunction but its role outside the central nervous system is insufficiently studied. Here, we identify a role of FMRP in liver disease. DESIGN: Mice lacking Fmr1 gene expression were used to study the role of FMRP during tumour necrosis factor (TNF)-induced liver damage in disease model systems. Liver damage and mechanistic studies were performed using real-time PCR, Western Blot, staining of tissue sections and clinical chemistry. RESULTS: Fmr1null mice exhibited increased liver damage during virus-mediated hepatitis following infection with the lymphocytic choriomeningitis virus. Exposure to TNF resulted in severe liver damage due to increased hepatocyte cell death. Consistently, we found increased caspase-8 and caspase-3 activation following TNF stimulation. Furthermore, we demonstrate FMRP to be critically important for regulating key molecules in TNF receptor 1 (TNFR1)-dependent apoptosis and necroptosis including CYLD, c-FLIPS and JNK, which contribute to prolonged RIPK1 expression. Accordingly, the RIPK1 inhibitor Necrostatin-1s could reduce liver cell death and alleviate liver damage in Fmr1null mice following TNF exposure. Consistently, FMRP-deficient mice developed increased pathology during acute cholestasis following bile duct ligation, which coincided with increased hepatic expression of RIPK1, RIPK3 and phosphorylation of MLKL. CONCLUSIONS: We show that FMRP plays a central role in the inhibition of TNF-mediated cell death during infection and liver disease.


Assuntos
Proteína do X Frágil de Retardo Mental/fisiologia , Hepatite Viral Animal/imunologia , Fator de Necrose Tumoral alfa/imunologia , Animais , Infecções por Arenaviridae/imunologia , Infecções por Arenaviridae/patologia , Linfócitos T CD8-Positivos/imunologia , Morte Celular/efeitos dos fármacos , Morte Celular/imunologia , Morte Celular/fisiologia , Células Cultivadas , Colestase/imunologia , Colestase/metabolismo , Colestase/patologia , Proteína do X Frágil de Retardo Mental/metabolismo , Hepatite Viral Animal/patologia , Hepatite Viral Animal/prevenção & controle , Hepatócitos/patologia , Imidazóis/farmacologia , Imidazóis/uso terapêutico , Indóis/farmacologia , Indóis/uso terapêutico , Vírus da Coriomeningite Linfocítica , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Serina-Treonina Quinases de Interação com Receptores/antagonistas & inibidores , Proteína Serina-Treonina Quinases de Interação com Receptores/fisiologia
3.
Int Endod J ; 53(1): 72-83, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31419325

RESUMO

AIM: To comparatively evaluate changes in the proliferation and mineralization abilities of dental pulp stem cells (DPSCs) from juvenile and adult rats in a lipopolysaccharide (LPS)-induced inflammatory microenvironment to provide a theoretical basis for the age-related differences observed in DPSCs during repair of inflammatory injuries. METHODOLOGY: DPSCs were isolated from juvenile (JDPSCs) and adult rats (ADPSCs), and senescence-associated ß-galactosidase staining was used to compare senescence between JDPSCs and ADPSCs. Effects of LPS on JDPSCs and ADPSCs proliferation were investigated by cell counting kit-8 assays and flow cytometry. Alizarin red staining, quantitative reverse transcription polymerase chain reaction and Western blot assay were used to examine the effects of LPS on mineralization-related genes and proteins in JDPSCs and ADPSCs. Immunohistochemistry was used to compare interleukin-1ß (IL-1ß) and osteocalcin (OCN) expression in the pulpitis model. Unpaired Student's t-tests and one-way anova were used for statistical analysis. RESULTS: DPSCs were isolated from juvenile and adult rat dental pulp tissues. At low concentrations (0.1-1 µg mL-1 ), LPS significantly promoted the proliferation of JDPSCs (P < 0.01) and ADPSCs (P < 0.01 or P < 0.05), with the effect being stronger in JDPSCs than in ADPSCs. In addition, mineralized nodules and the expression of mineralization-related genes (OCN, DSPP, ALP, BSP) increased significantly after stimulation with LPS (0.5 µg mL-1 ) in JDPSCs and ADPSCs (P < 0.01 or P < 0.05), and JDPSCs displayed a more obvious increase than ADPSCs. Western blots revealed OCN and ALP expression levels in JDPSCs treated with LPS were significantly upregulated (P < 0.05); meanwhile, ALP expression in ADPSCs increased slightly but significantly (P < 0.05), and OCN expression was not affected. Finally, IL-1ß expression was significantly higher (P < 0.05) and OCN expression was significantly lower (P < 0.05) in the inflamed dental pulp of adult rats than in juvenile rats. CONCLUSIONS: A certain degree of inflammatory stimulation promoted the proliferation and mineralization of DPSCs; however, this effect declined with age. The DPSCs of adult donors in an inflammatory microenvironment have a weaker repair ability than that of juvenile donors, who are better candidates for tissues damage repair.


Assuntos
Polpa Dentária , Células-Tronco , Afeto , Fosfatase Alcalina , Animais , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Humanos , Ratos
4.
APMIS ; 128(1): 10-19, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31642122

RESUMO

Atherogenesis is associated with chronic gut infections; however, the mechanisms are not clear. The aim of the study was to determine whether lipopolysaccharide of E. coli (E. coli LPS) may affect endothelial barrier and modify IL-10 expression in dendritic cells (DCs). Human umbilical vein endothelial cells (HUVECs) and monocyte-derived DCs were treated with E. coli LPS, apolipoprotein B100 (ApoB100) and 7-ketocholesterol (7-kCH) - harmful oxidized form of cholesterol. The effect of E. coli LPS, 7-kCH and ApoB100 on the barrier functions of HUVECs in real-time cell electric impedance sensing system (RTCA-DP) was assessed. Furthermore, the effect of 7-kCH and ApoB100 on barrier functions of HUVECs co-cultured with DCs previously treated with LPS was analyzed. Both E. coli LPS and 7-kCH decreased barrier functions of HUVECs and reduced tight junction protein mRNA expression, whereas ApoB100 increased endothelial barrier. In DCs, ApoB100 and E. coli LPS decreased IL-10 mRNA expression. In HUVECs co-cultured with DCs treated with LPS and subsequently pulsed with ApoB100 or 7-kCH, IL-10 mRNA expression was lower. E. coli LPS-exposed DCs diminished the protective effect of ApoB100 on endothelial integrity and led to the decrease in occludin mRNA expression. LPS potentially derived from gut microflora may destabilize endothelial barrier together with oxidized cholesterol and intensify the immunogenicity of ApoB100.


Assuntos
Células Dendríticas/efeitos dos fármacos , Escherichia coli/imunologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Interleucina-10/genética , Lipopolissacarídeos/imunologia , Junções Íntimas/efeitos dos fármacos , Apolipoproteína B-100/farmacologia , Células Cultivadas , Técnicas de Cocultura , Células Dendríticas/imunologia , Escherichia coli/química , Humanos , Cetocolesteróis/farmacologia , Lipopolissacarídeos/farmacologia , Ocludina/genética , Transdução de Sinais/efeitos dos fármacos , Proteínas de Junções Íntimas/genética
5.
Yi Chuan ; 41(12): 1119-1128, 2019 Dec 20.
Artigo em Chinês | MEDLINE | ID: mdl-31857283

RESUMO

Porcine skeletal muscle development is a complex biological process, and differentiation of skeletal muscle satellite cells is an important part of skeletal muscle development. In recent years, it has been found that lncRNA plays an important role in the differentiation of skeletal muscle satellite cells. Here we investigate the effect of lncRNA TCONS_00815878 on the differentiation of porcine skeletal muscle satellite cells. We first used qRT-PCR to detect the expression levels of TCONS_00815878 in six tissues (heart, spleen, lung, kidney, back muscles and leg muscles) of Yorkshire piglets within seven days of birth. At the same time, the expression levels of TCONS_00815878 at five different time points from the embryonic stage to the postnatal stage (35 d, 45 d, 55 d of embryos, and 7 d, 200 d of postpartum leg muscles) were examined. The expression of the differentiation marker genes MyoD, MyoG and MyHC was examined by knocking down TCONS_00815878 in porcine skeletal muscle satellite cells using antisense oligonucleotides (ASO). The target gene of TCONS_00815878 was predicted by bioinformatics analysis, and the function and pathway of its target gene were predicted online using DAVID software. The results showed that TCONS_00815878 had the highest expression level in pig myocardium and leg muscles. Within seven days after birth, TCONS_00815878 increased in the muscle tissue of pigs, and reached the peak of expression level on the 7th day. During the process of proliferation and differentiation of porcine skeletal muscle satellite cells, the expression level of TCONS_00815878 increased during the differentiation stage and peaked at 30 h of differentiation. After knocking down TCONS_00815878, the expression levels of MyoD, MyoG and MyHC were decreased, but the expression level of MyoD was significantly decreased (P<0.05). In addition, functional predictions revealed that the target gene of TCONS_00815878 is enriched in multiple biological processes, such as glycolysis and pyruvate metabolism, related to skeletal muscle satellite cell differentiation. This study speculates that lncRNA TCONS_00815878 may promote the differentiation of porcine skeletal muscle satellite cells.


Assuntos
Diferenciação Celular , Regulação da Expressão Gênica no Desenvolvimento , Músculo Esquelético , RNA Longo não Codificante , Células Satélites de Músculo Esquelético , Animais , Diferenciação Celular/genética , Proliferação de Células , Células Cultivadas , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Desenvolvimento Muscular , Músculo Esquelético/citologia , RNA Longo não Codificante/genética , Células Satélites de Músculo Esquelético/citologia , Suínos
6.
Zhonghua Yan Ke Za Zhi ; 55(12): 933-941, 2019 Dec 11.
Artigo em Chinês | MEDLINE | ID: mdl-31874508

RESUMO

Objective: To study the effects of human umbilical mesenchymal stem cells (HUMSCs) exosomes on the proliferation and apoptotic as well as migration of human retinal pigment epithelial cells (HRPE) in hypoxia, and explore its mechanism. Method: Direct adherent culture was adopted to cultivate umbilical cord mesenchymal stem cells and amplified to the fourth generation. Markers on the cell surface were identified by flow cytometry. Culture medium was collect without serum from the 4th generation umbilical cord mesenchymal stem cells. Exosomes were separated and extracted, then the ultrastructure was observed under electron microscope and examined expression of CD63 and CD9 protein by Western blot method with isolated and extracted exosomes. HRPE was cultivated in vitro culture, proliferation was detected at the time point of 0, 1, 2, 3, 4, 5 d with MTT assay under hypoxic condition. Meanwhile, the cell migration was quantified by Wound-Healing Assay under hypoxic condition at 0, 24, 48 and 72 h respectively combined with apoptosis test. The HRPE cells in the growth period were divided into 5 groups: the control group, the hypoxia group and the pretreated exosomes group (100, 200, 300 µg/ml). In all groups, apoptosis was observed by Annexin V/PI dual-dye flow cytometry after 48 h's incubation. Proliferation was observed by MTT assay and the migration was observed with Wound-Healing Assay. Results: Flow cytometry detection of the surface marker of HUMSCs in the 4th generation showed strong positive expression of CD105, CD73, CD90. It was suggested that HUMSCs with isolated culture had MSC specific phenotype with duction of lipids and osteoblasts in vitro. The separated exosomes were observed with spherical membranous structures in different sizes by scanning electron microscopy, and Western blot detected positive expression of CD63 and CD9. In vitro culture of HRPE detected by MTT assay for cell proliferation at the time of hypoxic 0, 1, 2, 3, 4, 5 d, the results showed that, comparing with time point 0 d, other groups had statistically significant OD values. In the first 2 days, the proliferation ability of RPE cells gradually increased as the time of hypoxia prolonged(1.862±0.135, 2.278±0.244). After 3 d, the proliferation ability of RPE cells gradually decreased(1.419±0.124, 1.599±0.156). Wound-Healing Assay results showed that the migration distance gradually increased as[(29.883±4.504), (36.200±1.928) µm] the time of hypoxia increased from 0 to 72 h. The cells were fully covering at the point of 72 h [(1.223±0.194), (0.430±0.299) µm]. Apoptosis test results showed that the number of apoptotic cells was different(3.628%±1.348%, 20.123%±1.183%) with the extension of hypoxia Oxygen before 2 d from 0 to 72 h. At the time of d3, there were more apoptotic cells(42.290%±3.217%). There is a significant difference from pre-2d.RPE cells were divided into 5 groups: the control group, the hypoxia group and the pretreated exosomes group (100, 200, 300 µg/ml).After 48 h hypoxia incubation, MTT assay results showed that, compared with the control group (1.870±0.499), the number of cell proliferation was significantly increased (t=-3.116, P<0.05), while compared with the hypoxia group(2.616±0.307), the proliferation number of exosomes was significantly reduced [(2.041±0.115), (1.931±0.205), (1.929±0.025); t=-4.920, -4.540, -5.286, P<0.01], and there was no significant difference between groups with different doses of the exosomes (F=1.181,P>0.05). Annexin V/PI dual-dye flow cytometry was used to observe the apoptosis results. Compared with the control group 1.180%±0.689%, the number of apoptosis in hypoxia group was significantly increased (19.273%±1.194%, t=-32.141, P<0.01), while compared with the hypoxia group, the number of apoptosis in the exosomes was significantly decreased (12.318%±1.087%, 11.878%±1.348%, 11.090%±1.716%; t=-10.547, -10.057, 9.589, P<0.01). There was no significant difference between the groups with different doses of exosomes (F=1.173, P>0.05). Wound-Healing Assay results showed that, compared with the control group(68.047±2.851) µm, the migration distance of the hypoxia group was significantly increased [(13.470±2.255)µm, t=36.778, P<0.01] while compared with the hypoxia group, the migration distance of the exosomes was reduced (33.110±1.774, 24.650±1.175, 26.440±1.674; t=11.766, 10.770, 11.311, P<0.01), and there was no significant difference between the groups of the exosomes (F=1.179, P>0.05). Conclusion: Human umbilical cord mesenchymal stem cells can effectively inhibit the apoptosis and migration of HRPE cells in hypoxia. It provides a theoretical basis for the research and treatment of RPE related diseases. (Chin J Ophthalmol, 2019, 55: 933-941).


Assuntos
Apoptose , Proliferação de Células , Exossomos , Hipóxia , Células-Tronco Mesenquimais , Retina , Células Cultivadas , Células Epiteliais , Humanos , Retina/citologia , Retina/metabolismo , Pigmentos da Retina , Cordão Umbilical
8.
Cell Physiol Biochem ; 53(6): 1029-1045, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31865646

RESUMO

BACKGROUND/AIMS: Fibro-adipogenic progenitors (FAPs), a muscle-resident stem cell population, have recently emerged as important actors of muscle regeneration by interacting with myogenic progenitors (MPs) to promote the formation of new muscle fibers. However, FAPs are also considered as main contributors of intramuscular fibrotic and fat depositions, resulting in a poor quality of muscles and a defective regeneration in aging and Duchenne Muscular Dystrophy disease (DMD). Therefore, the understanding of the control of FAP fate is an important aspect of muscle repair and homeostasis, but little is known in humans. We wondered the extent to which human FAP proliferation, adipogenesis and fibrogenesis can be regulated by human myogenic progenitors (MPs) in physiological and pathological contexts. METHODS: FAPs and MPs were isolated from skeletal muscles of healthy young or old donors and DMD patients. FAP/MP contact co-cultures and conditioned-media from undifferentiated MPs or differentiated myotubes were assessed on both proliferation and fibro-adipogenic differentiation of FAPs. RESULTS: We showed that soluble molecules released by MPs activate the phosphoinositide 3-kinase (PI3Kinase)/Akt pathway in FAPs, resulting in the stimulation of FAP proliferation. FAP differentiation was regulated by MP-derived myotubes through the secretion of pro-fibrogenic factors and anti-adipogenic factors. Importantly, the regulation of FAP adipogenic and fibrogenic fates by myotubes was found to be mediated by Smad2 phosphorylation and the gene expression of glioma-associated oncogene homolog 1 (GLI1). Surprisingly, the regulations of proliferation and differentiation were disrupted for FAPs and MPs derived from aged individuals and patients with DMD. CONCLUSION: Our results highlight a novel crosstalk between FAPs and the myogenic lineage in humans that could be crucial in the formation of adipocyte and myofibroblast accumulation in dystrophic and aged skeletal muscle.


Assuntos
Adipogenia , Desenvolvimento Muscular , Distrofia Muscular de Duchenne/fisiopatologia , Mioblastos/patologia , Adolescente , Adulto , Células-Tronco Adultas/patologia , Idoso , Envelhecimento , Células Cultivadas , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Adulto Jovem
9.
Zhongguo Gu Shang ; 32(11): 1026-1033, 2019 Nov 25.
Artigo em Chinês | MEDLINE | ID: mdl-31870051

RESUMO

OBJECTIVE: To construct and compare macrophage by gene modification of Wnt5a which co-cultured by human bone marrow-derived mesenchymal stem cells (MSCs) with two dimension (2D) and bone marrow aspirates in vitr transwell, in order to investigate the effect of Wnt5a signaling on cartilage homeostasis through regulation of macrophage pro-inflammatory responses. METHODS: Macrophages, MSCs and bone marrow aspirates specimens were extracted from 6 patients with severe knee deformities undergoing total knee arthroplasty(2 males, 4 females, aged from 58 to 71 years old) from September 2015 to December 2018. The synovial tissues of knee joints were exposed to type II collagenase and obtained single cell suspensions, and the purity of macrophages was determined by Ficoll gradient centrifugation and anti-CD14 antibody flow cytometry. The macrophages were transduced with IFN-γ combined with TNF-α for 48 h, and rAAV-lacZ or Wnt5a transfected for 24 h, then co-cultured model by human bone marrow-derived mesenchymal stem cells (MSCs) with two dimension (2D) and bone marrow aspirates in vitro transwell. HE staining, toluidine blue staining, X-gal staining and anti-Wnt5a, anti-II, X collagen immunohistochemical staining and enzyme-linked immunosorbent assay were applied to detect cell morphology and proliferation (cellularity), viral transfection efficiency respectively. RESULTS: Results of anti-CD68 immunohistochemistry showed macrophage of patients with osteoarthritis increased obviously. Anti-CD 14 flow cytometry confirmed that percentage of isolated synovial macrophages was 90.31%. The level of monocyte chemotactic protein-1 in supernatants was significantly increased after stimulation with IFN-γ and TNF-α, indicating that the macrophages were activated and at proinflammatory condition. After transduction with rAAV-lacZ for 3 days, X-gal staining indicated that lacZ gene transfer could efficiently transduce the activated macrophages with the efficiency over 97.50% for at least 21 days. After transfection macrophages by rAAV-Wnt5a stimulated expression of Wnt5a, and enhanced expression of Wnt5a between two models and inhibited the secretion of MCP-1. The expression of MCP-1 in Wnt5a group was 14.76 and 61.51 pg/ml respectively. In addition, rAAV-Wnt5a gene transfer could promote cell proliferation, chondrogenic differentiation and cartilage matrix synthesis. CONCLUSIONS: Under the condition of macrophage and MSCs 2D monolayer or aspirates transwe ll co-culture, rAAV-mediated over-expression of Wnt5a could promote maintenance of cartilage homeostasis and chondrogenic differentiation of MSCs via macrophage inflammatory response, macrophages may affect cartilage homeostasis and MSCs chondrogenesis through Wnt5a signaling pathway.


Assuntos
Condrogênese , Macrófagos , Idoso , Células da Medula Óssea , Diferenciação Celular , Células Cultivadas , Condrócitos , Técnicas de Cocultura , Feminino , Humanos , Inflamação , Masculino , Pessoa de Meia-Idade , Proteína Wnt-5a
10.
J Contemp Dent Pract ; 20(10): 1171-1178, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31883252

RESUMO

AIM: Aim of the study was to investigate the effect of PRP and MTA individually and combined on in vitro human bone marrow mesenchymal stem cells' (MSCs) proliferation and osteo/odontogenic differentiation potential. MATERIALS AND METHODS: MSCs were cultured in vitro with MTA, 5% PRP, 10% PRP, MTA with 5%PRP, and MTA with 10% PRP. Fetal calf serum (FCS) was used as control. Cell viability and proliferative efficiency were tested with cell adhesion and MTT assay. Osteo/odontogenic differentiation was assessed and quantified with alizarin red staining. RESULTS: MTA alone, MTA with 5% PRP, and MTA with 10% PRP showed significantly high proliferation at day 7 and 14 when compared to the control group. Enhanced differentiation and the highest calcium deposition was observed in MTA with the 10% PRP group. CONCLUSION: Within limitations of the in vitro environment, results imply an increased proliferation and induction of MSCs into osteo/odontogenic differentiation by the combination rather than a mere sealing of PRP by MTA. CLINICAL SIGNIFICANCE: PRP and MTA have the potential for true regeneration of the pulp tissue. Moreover, the combination of PRP and MTA can be utilized to expand the MSCs to generate adequate numbers for clinical applications, without xenogenic contamination. How to cite this article: Vanka A, Vishwakarma SK, Bhat MK, et al. Osteo/odontogenic Differentiation of Human Mesenchymal Stem Cells with Platelet-rich Plasma and Mineral Trioxide Aggregate. J Contemp Dent Pract 2019;20(10):1171-1178.


Assuntos
Células-Tronco Mesenquimais , Plasma Rico em Plaquetas , Compostos de Alumínio , Compostos de Cálcio , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Combinação de Medicamentos , Humanos , Óxidos , Silicatos
11.
Zhonghua Xin Xue Guan Bing Za Zhi ; 47(12): 993-999, 2019 Dec 24.
Artigo em Chinês | MEDLINE | ID: mdl-31877596

RESUMO

Objective: To investigate whether platelet-derived growth factor-BB (PDGF-BB) can regulate phenotypic transformation of pulmonary artery smooth muscle cells (PASMCs) via SIRT3 affecting glycolytic pathway. Methods: The PASMCs were isolated from Sprague Dawley rats. PASMCs were divided into 3 groups by using 2-deoxyglucose (2-DG), an inhibitor of the glycolytic pathway: normal control group, PDGF-BB group(30 ng/ml) and PDGF-BB (30 ng/ml)+2-DG (10 mmol/L) group. In lentivirus-mediated overexpression assay, cells were divided into control group, PDGF-BB group(30 ng/ml), PDGF-BB+deacetylase sirtuin-3 (SIRT3) overexpression group and PDGF-BB+empty vector group. The expression levels of phenotype related index such as α-smooth muscle actin (α-SMA), smooth muscle myosin heavy chain (SM-MHC), calponin, vimentin were detected by qRT-PCR and Western blot. Meanwhile, the expression of α-SMA was detected by cellular immunofluorescence staining. EDU staining was used to detect the proliferation of PASMCs. The expression of SIRT3 was detected by Western blot. The expressions of glucose transporter 1 and aerobic glycolytic enzymes were detected by qRT-PCR and Western blot in lentivirus-mediated overexpression assay. Results: (1) PDGF-BB affects PASMCs phenotypic transformation through glycolytic pathway: compared with normal control group, PDGF-BB significantly decreased the expressions of contractile phenotype markers such as α-SMA, SM-MHC, calponin mRNA and protein (all P<0.05), but it increased the expressions of the synthetic phenotype marker vimentin mRNA and protein (both P<0.05). Cellular immunofluorescence assay showed that PDGF-BB significantly decreased the number of α-SMA positive cells, while 2-DG reversed the process. (2) PDGF-BB promoted cell proliferation through glycolytic pathway: the proliferation of PASMCs was significantly higher in PDGF-BB group than in control group (P<0.05), and which could be significantly reduced by 2-DG (P<0.05). (3) PDGF-BB inhibited the expression of SIRT3 protein in PASMCs: the expression of SIRT3 protein in PDGF-BB group was lower than that in control group (P<0.05). (4) PDGF-BB affected glycolytic pathway through SIRT3:compared with the control group, PDGF-BB significantly increased the expression levels of glucose transporter 1 (Glut1), hexokinase 2 (HK2) and 6-phosphfructo-2-kinase 3 (PFKFB3) mRNA (all P<0.05), which was reserved by over-expression of SIRT3. There were no significant difference in mRNA expression levels between PDGF-BB group and PDGF-BB+empty vector group (P>0.05).Compared with the control group, PDGF-BB significantly increased the expression levels of Glut1, HK2 and PFKFB3 protein(all P<0.05), which was reserved by over-expression of SIRT3. There were no significant differences in protein expression levels between PDGF-BB group and PDGF-BB+empty vector group (all P>0.05). Conclusion: PDGF-BB regulates phenotypic transformation of PASMCs via SIRT3 affecting glycolytic pathway.


Assuntos
Becaplermina , Miócitos de Músculo Liso , Animais , Proliferação de Células , Células Cultivadas , Fenótipo , Proteínas Proto-Oncogênicas c-sis , Artéria Pulmonar , Ratos , Ratos Sprague-Dawley , Sirtuínas
12.
J Photochem Photobiol B ; 201: 111658, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31710923

RESUMO

The goal of the current experiment is to explore the influence of combined and/or single applications of red and near infrared (NIR) photobiomodulation (PBM) at different wavelengths, energy densities and times on cell viability, population doubling time (PDT), and apoptosis of in vitro cultures of human bone marrow-derived mesenchymal stem cells (hBM-MSCs) and h adipose-derived stem cells (hASCs). Both in vitro hBM-MSCs and hASCs were irradiated with 36 protocols using two different laser types (helium­neon [He-Ne] and diodes), four different laser wavelengths (HeNe laser, 630 nm, 810 nm, 630 + 810 nm); three different energy densities (0.6 J/cm2, 1.2 J/cm2, 2.4 J/cm2); and three different PBM times (1, 2, and 3). One-way ANOVA analysis showed that PBM with the 630 nm red laser significantly stimulated cellular viability of both hBM-MSCs and hASCs. The 630 nm red laser significantly decreased PDT of hBM-MSCs. One-way ANOVA demonstrated that the 630 + 810 laser significantly stimulated cellular viability, and significantly decreased PDT and apoptosis of hBM-MSCs and hASCs. Two-way ANOVA analysis showed that PBM with the 630 nm red laser and 630 + 810 nm laser significantly stimulated cellular viability of hASCs compared to the control hASCs, and experimental and control hBM-MSCs. Our study demonstrated that PBM with the combined 630 + 810 nm lasers significantly stimulated cell viability, and significantly decreased PDT and apoptosis of hBM-MSCs and hASCs in vitro. We reported new in vitro evidence where PBM administered at 630 nm (one and two times, 0.6 and 1.2 J/cm2) and 630 + 810 nm (three times, 2.4 J/cm2) significantly increased hASC cell viability compared to its control and the PBM-treated hBM-MSC groups.


Assuntos
Apoptose/efeitos da radiação , Lasers de Gás , Tecido Adiposo/citologia , Células da Medula Óssea/citologia , Sobrevivência Celular/efeitos da radiação , Células Cultivadas , Humanos , Terapia com Luz de Baixa Intensidade , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/efeitos da radiação , Células-Tronco/citologia , Células-Tronco/metabolismo , Células-Tronco/efeitos da radiação
13.
Biomed Khim ; 65(5): 361-365, 2019 Aug.
Artigo em Russo | MEDLINE | ID: mdl-31666406

RESUMO

In the model of induced neuronal resistance to the toxic effect of glutamate (deprivation of trophic factors), exosome secretion is demonstrated. Exosomes are secreted at the development of resistance during deprivation and at the first 24 h after preconditioning, as was shown by dot blot of extracellular fluid using anti-CD63 antibody. The autophagy inhibitor bafilomycin (0.01 µM) significantly reduces the quantity of the secreted exosomes at the stage of autophagy induction and at 24 h after induction. At the same time, inhibition of autophagy during the deprivation of trophic factors prevents the development of resistance, but inhibition of autophagy during the first 24 h after deprivation does not affect the development of resistance. We suggest that the long-term effects of preconditioning may be mediated by exosome secretion.


Assuntos
Autofagia , Exossomos/metabolismo , Neurônios/citologia , Células Cultivadas , Ácido Glutâmico , Humanos
14.
Biomed Khim ; 65(5): 366-373, 2019 Aug.
Artigo em Russo | MEDLINE | ID: mdl-31666407

RESUMO

Neurogenesis is a complex process which governs embryonic brain development and is importants for brain plasticity throughout the whole life. Postnatal neurogenesis occurs in neurogenic niches that regulate the processes of proliferation and differentiation of stem and progenitor cells under the action of stimuli that trigger the mechanisms of neuroplasticity. Cells of glial and endothelial origin are the key regulators of neurogenesis. It is known that physiological neurogeneses is crucial for memory formation, whereas reparative neurogenesis provides partial repair of altered brain structure and compensation of neurological deficits caused by brain injury. Dysregulation of neurogenesis is a characteristics of various neurodevelopmental and neurodegenerative diseases, particularly, Alzheimer's disease which is very important medical and social problem. In the in vitro model of the neurogenic niche using hippocampal neurospheres as a source of stem/progenitor cells and astrocytes, we studied effects of astrocyte activation on the expression of markers of different stages of cell proliferation and differentiation. We found that aberrant mechanisms of development of stem and progenitor cells, caused by the beta-amyloid (Aß1-42), can be partially restored by targeted activation of GFAP-expressing cells in the neurogenic niche.


Assuntos
Peptídeos beta-Amiloides/farmacologia , Astrócitos/citologia , Células-Tronco Neurais/citologia , Neurogênese , Fragmentos de Peptídeos/farmacologia , Diferenciação Celular , Células Cultivadas , Hipocampo/citologia , Humanos
15.
Biochemistry (Mosc) ; 84(9): 1047-1056, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31693464

RESUMO

Plant hormones produce cytotoxic effect on human cells and can trigger the processes unrelated to cell death, e.g., biosynthetic system stress. The goal of this study was to investigate activation of the endoplasmic reticulum (ER) stress by jasmonic acid (JA) and to distinguish between the responses of cultured immortalized non-tumorigenic HaCaT cells and epidermoid carcinoma A431 cells to this plant hormone. JA was used in the concentration of 2 mM, as it suppressed cell proliferation in both cell lines. We analyzed expression of genes associated with the activation of ER stress (GRP78, ATF4, CHOP), the structure of the ER and Golgi complex, and synthetic processes in the HaCaT and A431 cell lines. JA induced expression of genes responsible for the activation of ER stress and caused hypertrophic changes in the Golgi complex in both cell lines. However, the patterns of gene expression in the HaCaT and A431 cells were different, and higher levels of involucrin synthesis were observed in A431 but not in HaCaT cells, suggesting that JA activated differentiation of the tumor A431 cells only. Therefore, JA induced ER stress in both cell lines, but the consequences of ER stress were different for the epidermal immortalized non-tumorigenic and tumor cells.


Assuntos
Ciclopentanos/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Células Epidérmicas/efeitos dos fármacos , Células Epidérmicas/patologia , Neoplasias/patologia , Oxilipinas/farmacologia , Células Cultivadas , Relação Dose-Resposta a Droga , Células Epidérmicas/citologia , Humanos , Neoplasias/metabolismo , Relação Estrutura-Atividade
16.
Biochemistry (Mosc) ; 84(9): 1074-1084, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31693467

RESUMO

The CRISPR/Cas technology has a great potential in the treatment of many hereditary diseases. One of the prospective models for the CRISPR/Cas-mediated therapy is spinal muscular atrophy (SMA), a disease caused by deletion of the SMN1 gene that encodes the SMN protein required for the survival of motor neurons. SMA patients' genomes contain either single or several copies of SMN2 gene, which is a paralog of SMN1. Exon 7 of SMN2 has the single-nucleotide substitution c.840C>T leading to the defective splicing and decrease in the amounts of the full-length SMN. The objective of this study was to create and test gene-editing systems for correction of the single-nucleotide substitution c.840C>T in exon 7 of the SMN2 gene in fibroblasts, induced pluripotent stem cells, and motor neuron progenitors derived from a SMA patient. For this purpose, we used plasmid vectors expressing CRISPR/Cas9 and CRISPR/Cpf1, plasmid donor, and 90-nt single-stranded oligonucleotide templates that were delivered to the target cells by electroporation. Although sgRNA_T2 and sgRNA_T3 guiding RNAs were more efficient than sgRNA_T1 in fibroblasts (p < 0.05), no significant differences in the editing efficiency of sgRNA_T1, sgRNA_T2, and sgRNA_T3 was observed in patient-specific induced pluripotent stem cells and motor neuron progenitors. The highest editing efficiency in induced pluripotent stem cells and motor neuron progenitors was demonstrated by the sgRNA_T1 and 90-nt single-stranded oligonucleotide donors.


Assuntos
Sistemas CRISPR-Cas , Éxons/genética , Polimorfismo de Nucleotídeo Único/genética , Sistemas CRISPR-Cas/genética , Células Cultivadas , Humanos , Proteína 2 de Sobrevivência do Neurônio Motor/genética
17.
Zhongguo Yi Xue Ke Xue Yuan Xue Bao ; 41(5): 589-594, 2019 Oct 30.
Artigo em Chinês | MEDLINE | ID: mdl-31699187

RESUMO

Objective To investigate the effect of microRNA-133b(miR-133b)on cardiac fibrosis and its mechanism.Methods Human cardiac fibroblasts(CFs)were harvested.The proliferation of CFs was detected by CCK8 during the overexpression and knock-down of miR-133b.The expressions of connective tissue growth factor(CTGF),α-smooth muscle actin(α-SMA),collagen Ⅰ,and collagen Ⅲ were detected with qRT-PCR and Western blot analysis after miR-133b overexpression or downexpression.Target genes of miR-133b were predicted by bioinformatics software.Dual-luciferase activity assay were used to verify a target gene of miR-133b.Results qRT-PCR showed that the expression level of miR-133b in the miR-133b mimic group was significantly higher than that in the negative control group(t=26.219,P=0.000).The expression level of miR-133b in the miR-133b inhibitor group was significantly lower than that in the negative control group(t=6.738,P=0.003).After 21,45,69,93,and 117 hours of transfection,the proliferation ability of CFs significantly decreased in the miR-133b mimic group but significantly increased in the miR-133b group(all P<0.05,compared with the negative control group).After overexpression of miR-133b,the mRNA and protein levels of CTGF(t=9.213,P=0.001;t=8.195,P=0.001),α-SMA(t=6.511,P =0.003;t=4.434,P=0.011),collagenⅠ(t=3.172,P=0.034;t=4.053,P=0.015)and collagen Ⅲ(t=6.404,P=0.003;t=5.319,P=0.006)were significantly down-regulated.After the expression of miR-133b was knocked down,the mRNA and protein levels of CTGF(t=9.439,P=0.001;t=14.100,P=0.000),α-SMA(t=4.519,P=0.011;t=4.377,P=0.012),collagen Ⅰ(t=5.966,P=0.004;t=5.514,P=0.005)and collagen Ⅲ(t=4.622,P=0.010;t=4.996,P=0.008)were significantly increased.The relative luciferase activity of the cells co-transfected with miR-133b mimic and WT 3'UTR expression vector was significantly lower than that of the cells co-transfected with mimic control and WT 3'UTR expression vectors(t=5.654,P=0.005);however,there was no significant difference in relative luciferase activity between cells co-transfected with miR-133b mimic and MUT 3'UTR expression vectors and cells co-transfected with mimic control and MUT 3'UTR expression vectors(t=0.380,P=0.724).Conclusion miR-133b may affect the activation and proliferation of CFs by targeting CTGF and thus improve cardiac fibrosis.


Assuntos
Fibroblastos/citologia , MicroRNAs/genética , Miocárdio/patologia , Actinas/metabolismo , Proliferação de Células , Células Cultivadas , Colágeno/metabolismo , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Fibrose , Humanos
18.
Zhongguo Yi Xue Ke Xue Yuan Xue Bao ; 41(5): 595-600, 2019 Oct 30.
Artigo em Chinês | MEDLINE | ID: mdl-31699188

RESUMO

Objective To explore the role of multidrug resistance gene-1(MDR1)gene in methotrexate(MTX)resistance in patients with rheumatoid arthritis(RA).Methods Fibroblast-like synoviocytes(FLS)from RA patients were infected with recombinant adenovirus Ad-EGFP-MDR1 in vitro to obtain MDR1 over-expressed RA FLS.The transcription level of MDR1 gene and the expression level of its coding product P-glycoprotein(P-gp) rotein were detected by real-time PCR and Western blot analysis.The efflux function was verified by rhodamine 123 efflux assay.The resistance to MTX was detected by MTT assay.Results RA FLS were infected with recombinant adenovirus Ad-EGFP-MDR1;72 hours later,the particles size in MDR1 over-expressed RA FLS increased,the cell volume became larger,and the growth rate decreased.The transcription level of MDR1(1.4325±0.3924 vs.0.0650±0.0070;t=6.035,P=0.004),the expression level of P-gp protein(1.8667±0.2857 vs. 0.9367±0.0551;t=5.536,P=0.005),and the ability of extracellular rhodamine 123(979.43±196.81 vs.1680.06±147.04;t=-4.940,P=0.008) in MDR1 over-expressed RA FLS were significantly higher than those of negative virus control RA-FLS,and the survival rate of MDR1 over-expressed RA FLS was significantly increased at each concentration of MTX(P<0.05).Conclusion The high expression of MDR1 can affect the efflux ability to MTX by up-regulating the expression of P-gp,thus enhancing the drug resistance to MTX in RA FLS.


Assuntos
Artrite Reumatoide/tratamento farmacológico , Resistência a Medicamentos , Fibroblastos/efeitos dos fármacos , Metotrexato/farmacologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Artrite Reumatoide/genética , Células Cultivadas , Humanos , Membrana Sinovial/citologia
19.
Cell Physiol Biochem ; 53(5): 832-850, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31703162

RESUMO

BACKGROUND/AIMS: Runt-related transcription factor 2 (Runx2) is a master regulator of osteogenic differentiation, but most of the direct downstream targets of RUNX2 during osteogenesis are unknown. Likewise, High-temperature requirement factor A1 (HTRA1) is a serine protease expressed in bone, yet the role of Htra1 during osteoblast differentiation remains elusive. We investigated the role of Htra1 in osteogenic differentiation and the transcriptional regulation of Htra1 by RUNX2 in primary mouse mesenchymal progenitor cells. METHODS: Overexpression of Htra1 was carried out in primary mouse mesenchymal progenitor cells to evaluate the extent of osteoblast differentiation. Streptavidin agarose pulldown assay, chromatin immunoprecipitation assay, and dual luciferase assay were carried out to investigate the interaction of RUNX2 protein at the Htra1 promoter during osteoblast differentiation. RESULTS: Overexpression of Htra1 increased the production of mineralized bone matrix, upregulating several osteoblast genes, such as Sp7 transcription factor (Sp7) and Alkaline phosphatase, liver/bone/kidney (Alpl). In addition, Htra1 upregulated osteogenesis-related signalling genes, such as Fibroblast growth factor 9 (Fgf9) and Vascular endothelial growth factor A (Vegfa). A series of experiments confirmed Htra1 as a direct RUNX2 transcriptional target. Overexpression of Runx2 resulted in the upregulation of Htra1 mRNA and protein. Chromatin immunoprecipitation and streptavidin agarose pull-down assays showed that RUNX2 binds a proximal -400 bp region of the Htra1 promoter during osteogenic differentiation. Dual luciferase assays confirmed that RUNX2 activates the proximal Htra1 promoter during osteogenic differentiation. Mutation of putative RUNX2 binding sites revealed that RUNX2 interacts with the Htra1 promoter at -252 bp and -84 bp to induce Htra1 expression. CONCLUSION: We demonstrate that Htra1 is a positive regulator of osteogenic differentiation, showing for the first time that Htra1 is a direct downstream target of RUNX2.


Assuntos
Diferenciação Celular , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Serina Peptidase 1 de Requerimento de Alta Temperatura A/metabolismo , Animais , Sítios de Ligação , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Fator 9 de Crescimento de Fibroblastos/metabolismo , Serina Peptidase 1 de Requerimento de Alta Temperatura A/genética , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Osteogênese , Regiões Promotoras Genéticas , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular/metabolismo
20.
Hua Xi Kou Qiang Yi Xue Za Zhi ; 37(5): 469-475, 2019 Oct 01.
Artigo em Chinês | MEDLINE | ID: mdl-31721491

RESUMO

OBJECTIVE: This study aims to compare the osteogenic differentiation capability of stem cells derived from human inflammatory periodontal ligament tissues (iPDLSCs) with those of stem cells derived from healthy periodontal ligament tissues (hPDLSCs). Both types of tissues were induced by stromal cell derived factor (SDF-1) in vitro. METHODS: iPDLSCs and hPDLSCs were primarily cultured by tissue digestion method and purified by limited dilution cloning. The cells were passaged and identified by stem cell surface marker expression through flow cytometry. Then, we used thiazolyl blue tetrazolium bromide to detect and compare the proliferation capabilities of the iPDLSCs and hPDLSCs. Express of bone volumes were detected by alizarin red staining after SDF-1 was added to the cells. Using alkaline phosphatase, we evaluated the osteogenic differentiation capability of the cells induced by SDF-1. The expression levels of the osteogenesis-related genes of the cells induced by SDF-1 were determined by reverse transcription-polymerase chain reaction. RESULTS: After purification, both iPDLSCs and hPDLSCs expressed stem cell markers. hPDLCSs had a higher proliferation capability than iPDLSCs. Osteogenesis-related genes had higher expression levels in the cells induced by SDF-1 than in those without induction (P<0.05). SDF-1 at 50 and 200 ng·mL⁻¹ concentration greatly affected the differen-tiation capabilities of iPDLSCs and hPDLSCs respectively. CONCLUSIONS: iPDLSCs and hPDLSCs had osteogenic differentia-tion capability. The level of osteogenic differentiation in normal and inflamed periodontal ligament stem cells increases after SDF-1 induction.


Assuntos
Osteogênese , Ligamento Periodontal , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Humanos , Células-Tronco , Células Estromais
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