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Emerging evidence suggests the dysregulation of long non-coding RNAs (lncRNAs) involved in pancreatic cancer (PC). However, the function of LINC00930 in PC has not been elaborated. In this study, we found that LINC00930 was significantly down-regulated in PC cell lines and tissues, and associated with tumor size, lymphatic metastasis, TNM stage and poor prognosis. According to the bioinformatics database, the downregulation of LINC00930 was a common event in PC associated with prognosis and EMT. Overexpression of LINC00930 inhibited the aggressive cancer phenotypes including proliferation, metastasis and epithelial-mesenchymal transition (EMT) of PC in vitro and in vivo. Bioinformatics and dual-luciferase reporter assay indicated that miR-6792-3p could directly bind to LINC00930. Additionally, the Zinc finger and BTB domain containing 16 (ZBTB16) was significantly declined in PC, which was predicted to be the downstream gene of miR-6792-3p. MiR-6792-3p mimic rescued the decreased proliferation, metastasis and EMT caused by ZBTB16 in PC cells. The LINC00930/miR-6792-3p/ZBTB16 axis was associated with the malignant progression and process of PC. The relative expression of LINC00930 was negatively correlated with the expression of miR-6792-3p and was closely linked with ZBTB16 levels in PC. LINC00930 might serve as a potential prognostic biomarker and therapeutic target for PC.
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Proliferação de Células , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , MicroRNAs , Neoplasias Pancreáticas , RNA Longo não Codificante , Humanos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Transição Epitelial-Mesenquimal/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Proliferação de Células/genética , Animais , Camundongos , Linhagem Celular Tumoral , Masculino , Feminino , Prognóstico , Pessoa de Meia-Idade , Movimento Celular/genéticaRESUMO
An Ag-catalysed three-component reaction of alkynyl aryl ketones bearing an ortho-methoxy group, element selenium, and arylboronic acid, providing a facile route to selenofunctionalized chromone products has been developed. This protocol features high efficiency and high regioselectivity, and the use of selenium powder as the selenium source. Mechanistic experiments indicated that the combined oxidative effect of (bis(trifluoroacetoxy)iodo)benzene and oxygen in the air pushes the catalytic redox cycle of the Ag catalyst and the phenylselenium trifluoroacetate formed in situ is the key intermediate of the PIFA-mediated 6-endo-electrophilic cyclization and selenofunctionalization reaction of alkynyl aryl ketones.
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Cetonas , Selênio , Ácidos Borônicos , Ciclização , PrataRESUMO
Theranostic agents for concurrent cancer therapy and diagnosis have begun attracting attention as a promising modality. However, accurate imaging and identification remains a great challenge for theranostic agents. Here, we designed and synthesized a novel theranostic agent H6M based on the "double-locked" strategy by introducing an electron-withdrawing nitro group into 1-position of a pH-responsive 3-amino-ß-carboline and further covalently linking the hydroxamic acid group, a zinc-binding group (ZBG), to the 3-position of ß-carboline to obtain histone deacetylase (HDAC) inhibitory effect for combined HDAC-targeted therapy. We found that H6M can be specifically reduced under overexpressed nitroreductase (NTR) to produce H6AQ, which emits bright fluorescence at low pH. Notably, H6M demonstrated a selective fluorescence imaging via successive reactions with NTR (first "key") and pH (second "key"), and precisely identified tumor margins with a high S/N ratio to guide tumor resection. Finally, H6M exerted robust HDAC1/cancer cell inhibitory activities compared with a known HDAC inhibitor SAHA. Therefore, the NTR/pH-activated theranostic agent provided a novel tool for precise diagnosis and efficient tumor therapy.
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Antineoplásicos/farmacologia , Inibidores de Histona Desacetilases/química , Inibidores de Histona Desacetilases/farmacologia , Medicina de Precisão/métodos , Animais , Antineoplásicos/química , Linhagem Celular Tumoral , Feminino , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Camundongos Endogâmicos BALB C , Neoplasias Experimentais/diagnóstico por imagem , Neoplasias Experimentais/cirurgia , Neoplasias Experimentais/terapia , Nitrorredutases/metabolismo , Ratos , Espectrometria de Fluorescência , Cirurgia Assistida por Computador , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Whole-organ decellularization has been identified as a promising choice for tissue engineering. The aim of the present study was to engineer intact whole rat liver scaffolds and repopulate them with hepatocytes and endothelial progenitor cells (EPCs) in a bioreactor. Decellularized liver scaffolds were obtained by perfusing Triton X-100 with ammonium hydroxide. The architecture and composition of the original extracellular matrix were preserved, as confirmed by morphologic, histological, and immunolabeling methods. To determine biocompatibility, the scaffold was embedded in the subcutaneous adipose layer of the back of a heterologous animal to observe the infiltration of inflammatory cells. Hepatocytes were reseeded using a parenchymal injection method and cultured by continuous perfusion. EPCs were reseeded using a portal vein infusion method. Morphologic and functional examination showed that the hepatocytes and EPCs grew well in the scaffold. The present study describes an effective method of decellularization and recellularization of rat livers, providing the foundation for liver engineering and the development of bioartificial livers.
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Células Progenitoras Endoteliais/transplante , Matriz Extracelular/química , Hepatócitos/transplante , Fígado/citologia , Engenharia Tecidual/métodos , Alicerces Teciduais/química , Animais , Reatores Biológicos , Proliferação de Células , Células Cultivadas , Células Progenitoras Endoteliais/citologia , Hepatócitos/citologia , Fígado/química , Fígado/ultraestrutura , Camundongos Endogâmicos C57BL , Ratos , Ratos Sprague-DawleyRESUMO
T1D is an autoimmune disease, which may be caused by lack of insulin-secreting ß cells due to damage of autoimmune system. Living with T1D is a challenge for the child and the family; cell transplantation is a treatment option for diabetes in children. To establish a microenvironment suitable for cell growth and proliferation as well as for sustained cellular function, we used MIN-6 ß cells as seed cells and SF-IV collagen as a 3D composite scaffold to construct artificial pancreas in this experiment. The cell viabilities were determined by MTT assay, and the response of cells to different glucose concentrations was observed by glucose stimulation test. Artificial pancreas was transplanted into the abdominal cavity of T1D mice, and the changes of blood glucose were monitored. After 10 days, insulin expression was detected by immunohistochemical method, and the claybank stained area showed effectiveness of insulin secretion. A series of experiments showed that implantation of 3D cell scaffold into the abdominal cavity can effectively control the blood glucose level of T1D mice. It also had longer-lasting hypoglycemic effects than simple cell transplantation, which was expected to become a new method for the treatment of T1D.
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Técnicas de Cultura de Células , Técnicas de Cocultura , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Tipo 1/sangue , Células Secretoras de Insulina/citologia , Animais , Glicemia/análise , Linhagem Celular , Proliferação de Células , Sobrevivência Celular , Feminino , Imuno-Histoquímica , Insulina/sangue , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND: Pancreatic cancer is one of the most aggressive cancers, and the aggressiveness of pancreatic cancer is in part due to its intrinsic and extrinsic drug resistance characteristics, which are also associated with the acquisition of epithelial-to-mesenchymal transition (EMT). Increasing evidence suggests that EMT-type cells share many biological characteristics with cancer stem-like cells. And miR-200 has been identified as a powerful regulator of EMT. METHODS: Cancer Stem Cells (CSCs) of human pancreatic cancer cell line PANC-1 were processed for CD24, CD44 and ESA multi-colorstaining, and sorted out on a BD FACS Aria II machine. RT-qPCR was performed using the miScript PCR Kit to assay the expression of miR-200 family. In order to find the role of miR-200a in the process of EMT, miR-200a mimic was transfected to CSCs. RESULTS: Pancreatic cancer cells with EMT phenotype displayed stem-like cell features characterized by the expression of cell surface markers CD24, CD44 and epithelial-specific antigen (ESA), which was associated with decreased expression of miR-200a. Moreover, overexpression of miR-200a was resulted in down-regulation of N-cadherin, ZEB1 and vimentin, but up-regulation of E-cadherin. In addition, miR-200a overexpression inhibited cell migration and invasion in CSCs. CONCLUSION: In our study, we found that miR-200a played an important role in linking the characteristics of cancer stem-like cells with EMT-like cell signatures in pancreatic cancer. Selective elimination of cancer stem-like cells by reversing the EMT phenotype to mesenchymal-to-epithelial transition (MET) phenotype using novel agents would be useful for prevention and/or treatment of pancreatic cancer.
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Transição Epitelial-Mesenquimal/fisiologia , MicroRNAs/biossíntese , Células-Tronco Neoplásicas/metabolismo , Neoplasias Pancreáticas/metabolismo , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Citometria de Fluxo/métodos , Humanos , Células-Tronco Neoplásicas/patologia , Neoplasias Pancreáticas/patologiaRESUMO
Interactions between cells are of fundamental importance in affecting cell function. In vivo, endothelial cells and islet cells are close to each other, which makes endothelial cells essential for islet cell development and maintenance of islet cell function. We used endothelial cells to construct 3D pseudo-islets, which demonstrated better glucose regulation and greater insulin secretion compared to conventional pseudo-islets in both in vivo and in vitro trials. However, the underlying mechanism of how endothelial cells promote beta cell function localized within islets is still unknown. We performed transcriptomic sequencing, differential gene analysis, and enrichment analysis on two types of pseudo-islets to show that endothelial cells can promote the function of internal beta cells in pseudo-islets through the BTC-EGFR-JAK/STAT signaling pathway. Min6 cells secreted additional BTC after co-culture of endothelial cells with MIN6 cells outside the body. After BTC knockout in vitro, we found that beta cells functioned differently: insulin secretion levels decreased significantly, while the expression of key proteins in the EGFR-mediated JAK/STAT signaling pathway simultaneously decreased, further confirming our results. Through our experiments, we elucidate the molecular mechanisms by which endothelial cells maintain islet function in vitro, which provides a theoretical basis for the construction of pseudo-islets and islet cell transplants for the treatment of diabetes mellitus.
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Células Endoteliais , Transdução de Sinais , Animais , Camundongos , Linhagem Celular , Células Endoteliais/metabolismo , Receptores ErbB/metabolismo , Receptores ErbB/genética , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Janus Quinases/metabolismo , Fatores de Transcrição STAT/metabolismoRESUMO
The tumor microenvironment (TME) of pancreatic ductal adenocarcinoma (PDAC) is characterized by deposition of desmoplastic matrix (including collagen and hyaluronic acid). And the interactions between tumor-associated macrophages (TAMs) and tumor cells play a crucial role in progression of PDAC. Hence, the appropriate model of tumor cell-macrophage interaction within the unique PDAC TME is of significantly important. To this end, a 3D tumor niche based on dual-crosslinking gelatin methacrylate and hyaluronic acid methacrylate hydrogels was constructed to simulate the desmoplastic tumor matrix with matching compressive modulus and composition. The bionic 3D tumor niche creates an immunosuppressive microenvironment characterized by the downregulation of M1 markers and upregulation of M2 markers in TAMs. Mechanistically, RNA-seq analysis revealed that the PI3K-AKT signaling pathway might modulate the phenotypic balance and recruitment of macrophages through regulating SELE and VCAM-1. Furthermore, GO and GSEA revealed the biological process of leukocyte migration and the activation of cytokine-associated signaling were involved. Finally, the 3D tumor-macrophage niches with three different ratios were fabricated which displayed increased M2-like polarization and stemness. The utilization of the 3D tumor niche has the potential to provide a more accurate investigation of the interplay between PDAC tumor cells and macrophages within an in vivo setting.
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Carcinoma Ductal Pancreático , Gelatina , Ácido Hialurônico , Metacrilatos , Microambiente Tumoral , Macrófagos Associados a Tumor , Gelatina/química , Ácido Hialurônico/química , Ácido Hialurônico/farmacologia , Humanos , Macrófagos Associados a Tumor/metabolismo , Macrófagos Associados a Tumor/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacos , Carcinoma Ductal Pancreático/patologia , Carcinoma Ductal Pancreático/metabolismo , Metacrilatos/química , Metacrilatos/farmacologia , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/metabolismo , Hidrogéis/química , Linhagem Celular Tumoral , Materiais Biomiméticos/química , Materiais Biomiméticos/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
Delta-like ligand 3 (DLL3) is overexpressed in small-cell lung cancer (SCLC) and has been considered an attractive target for SCLC therapy. Rovalpituzumab tesirine (Rova-T) was the first DLL3-targeted antibody-drug conjugate (ADC) to enter clinical studies. However, serious adverse events limited progress in the treatment of SCLC with Rova-T. In this study, we developed a novel DLL-3-targeted ADC, FZ-AD005, by using DXd with potent cytotoxicity and a relatively better safety profile to maximize the therapeutic index. FZ-AD005 was generated by a novel anti-DLL3 antibody FZ-A038 and a valine-alanine (Val-Ala) dipeptide linker to conjugate DXd. Moreover, Fc-silencing technology was introduced in FZ-AD005 to avoid off-target toxicity mediated by FcγRs and showed negligible Fc-mediated effector functions in vitro. In preclinical evaluation, FZ-AD005 exhibited DLL3-specific binding and demonstrated efficient internalization, bystander killing, and excellent in vivo antitumor activities in cell line-derived xenografts (CDX) and patient-derived xenograft (PDX) models. FZ-AD005 was stable in circulation with acceptable pharmacokinetic profiles in cynomolgus monkeys. FZ-AD005 was well tolerated in rats and monkeys. The safety profile of FZ-AD005 was favorable and the highest non-severely toxic dose was 30 mg/kg in cynomolgus monkeys. In conclusion, FZ-AD005 has the potential to be a superior DLL3-targeted ADC with a wide therapeutic window and is expected to provide clinical benefits for the treatment of SCLC patients.
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Chronic hepatitis B virus infection is the dominant global cause of hepatocellular carcinoma (HCC), especially hepatitis B virus-X (HBx) plays a major role in this process. HBx protein promotes cell cycle progression, inactivates negative growth regulators, and binds to and inhibits the expression of p53 tumor suppressor gene and other tumor suppressor genes and senescence-related factors. However, the relationship between HBx and autophagy during the HCC development is poorly known. Previous studies found that autophagy functions as a survival mechanism in liver cancer cells. We suggest that autophagy plays a possible role in the pathogenesis of HBx-induced HCC. The present study showed that HBx transfection brought about an increase in the formation of autophagosomes and autolysosomes. Microtubule-associated protein light chain 3, Beclin 1, and lysosome-associated membrane protein 2a were up-regulated after HBx transfection. HBx-induced increase in the autophagic level was increased by mTOR inhibitor rapamycin and was blocked by treatment with the PI3K-Akt inhibitor LY294002. The same results can also be found in HepG2.2.15 cells. These results suggest that HBx activates the autophagic lysosome pathway in HepG-2 cells through the PI3K-Akt-mTOR pathway.
Assuntos
Autofagia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Transativadores/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Família da Proteína 8 Relacionada à Autofagia , Proteína Beclina-1 , Cromonas/farmacologia , Regulação para Baixo/efeitos dos fármacos , Expressão Gênica , Células Hep G2 , Vírus da Hepatite B/fisiologia , Interações Hospedeiro-Patógeno , Humanos , Lisossomos/metabolismo , Lisossomos/patologia , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Microscopia Eletrônica de Transmissão , Morfolinas/farmacologia , Fagossomos/metabolismo , Fagossomos/patologia , Fosfatidilinositol 3-Quinases/genética , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas c-akt/genética , Transdução de Sinais , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/genética , Transativadores/biossíntese , Transativadores/genética , Ativação Transcricional , Proteínas Virais Reguladoras e AcessóriasRESUMO
OBJECTIVE: To investigate the role of autophagy in the injury of HepG-2 cells induced by hepatitis B virus x protein (HBx). METHODS: After HBx transfection, the cells were used to detect the formation of autophagosomes and observed by transmission electron microscopy, monodansylcadaverine (MDC) staining autophagic vacuole (AV), immunofluorescent ce staining microtubule-associated protein light chain 3 ( MAP1-LC3 ) protein, and Western blotting examining the ratio of LC3-II/LC3-I (gray level: 0.760 ± 0.078 vs 0.520 ± 0.086, P < 0.05), beclin 1 (gray level: 0.875 ± 0.093 vs 0.220 ± 0.087, P < 0.05)and lysosome associated membrane protein 2a ( lamp2a ) protein (gray level: 0.320 ± 0.061 vs 0.120 ± 0.064, P < 0.05) levels. RESULTS: (1) HBx transfected upregulated the expression of LC3-II, LC3-I, beclin 1 and lamp2a protein. (2) HBx transfected brought about an increase in the formation of autophagosomes and autolysosomes. CONCLUSION: HBx activates the autophagic lysosome pathway in HepG-2 cells through the LC3/beclin1 pathway.
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Autofagia , Neoplasias Hepáticas/patologia , Transativadores/genética , Células Hep G2 , Humanos , Neoplasias Hepáticas/metabolismo , Transfecção , Proteínas Virais Reguladoras e AcessóriasRESUMO
BACKGROUND: Pancreatic cancer (PC) is highly malignant and difficult to detect, while few blood markers are currently available for diagnosing PC. METHODS: We obtained differential expression genes (DEGs) from GEO (gene expression omnibus) database and assessed by quantitative real-time polymerase chain reaction (qRT-PCR), receiver operating characteristic (ROC), univariate and multifactorial regression analysis, and survival analysis in our clinic center. Through the TCGA (the cancer genome atlas) database, we analyzed functional enrichment, different risk groups with survival analysis, immunological features, and the risk score established by the Cox regression model and constructed a nomogram. RESULT: Immunoglobulin heavy constant delta (IGHD) was remarkably upregulated in peripheral blood from PC patients, and IGHD was a potential independent biomarker for PC diagnosis (ROC sensitivity, 76.0%; specificity, 74.2%; area under the curve (AUC) = 0.817; univariate logistic regression analysis: odds ratio (OR) 1.488; 95% confidence interval (CI) 1.182-1.872; P < 0.001; multiple logistic: OR 2.097; 95% CI 1.276-3.389, P = 0.003). In addition, the IGHD expression was remarkably reduced after resectioning the primary tumor. High IGHD expression indicated higher lymphocyte infiltration and increased activities of immunological pathways in PC patients. KRAS and SMAD were observed with a prominent difference among top mutated genes between the two groups. The risk score predicted reliable clinical prognosis and drug responses. Furthermore, a nomogram with the risk score and clinical characteristics was constructed, showing a better predictive performance. CONCLUSION: IGHD is a valuable PC diagnosis, prognosis, and therapeutic response marker.
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Antibody drug conjugates (ADCs) have emerged as a highly promising class of cancer therapeutics, comprising antibodies, effector molecules, and linkers. Among them, DS-8201a with DXd as the effector molecule, has shown remarkable anti-tumor efficacy against solid tumors, sparking a surge of interest in ADCs with camptothecin derivatives as ADC effector molecules. In this study, we introduced and successfully constructed quaternary ammonium ADCs utilizing camptothecin derivatives WL-14 and CPTS-1 for the first time. All four ADCs displayed excellent stability under physiological conditions and in plasma, facilitating their prolonged circulation in vivo. Moreover, the four ADCs, employing Val-Cit or Val-Ala dipeptide linkers effectively achieved complete release of the effector molecules via cathepsin B. Although, the in vitro antitumor activity of these ADCs was comparatively limited, the development of quaternary ammonium ADCs based on novel camptothecin derivatives as effector molecules is still a viable and promising strategy. Significantly, our study provides valuable insights into the crucial role of linker optimization in ADCs design.
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Compostos de Amônio , Antineoplásicos , Imunoconjugados , Neoplasias , Humanos , Camptotecina , Imunoconjugados/química , Anticorpos/uso terapêutico , Neoplasias/tratamento farmacológico , Antineoplásicos/uso terapêutico , Linhagem Celular TumoralRESUMO
Splenic hemorrhagic shock is a typical emergency in surgery, seriously threatening human beings' life. Emerging evidence shows that microRNAs (miRNAs) are closely related to inflammation and immunity in the body. However, the detailed effects and underlying mechanisms of miRNAs on the immune function of splenic hemorrhagic shock have not been revealed yet. In the present study, we construct the rat hemorrhagic shock model, and the rats are further cured with splenic blood transport clipping recanalization (SBTCR). MiR-18b-5p was highly expressed in the spleen of hemorrhagic shock rats detected by the qRT-PCR assay. Functionally, down-regulation of miR-18b-5p notably inhibited the levels of SOD1, iNOS and IL-6 in macrophages isolated from splenic tissues detected by qRT-PCR and ELISA assays. In addition, inhibition of miR-18b-5p significantly decreased the M1/M2 ratio of macrophages. Besides, knockdown of miR-18b-5p obviously reduced the Th1/Th2 ratio of CD4+ T cells. Moreover, HIF-1α was predicted as a target gene of miR-18b-5p, which was further confirmed by dual-luciferase reporter assay, and HIF-1α was negatively associated with miR-18b-5p. Furthermore, overexpression of HIF-1α partially restored the effects of miR-18b-3p on inflammation and immunity in macrophages. Taken together, miR-18b-5p may be a novel therapeutic candidate target in splenic hemorrhagic shock treatment.
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MicroRNAs , Choque Hemorrágico , Baço , Animais , Proliferação de Células , Regulação para Baixo , Subunidade alfa do Fator 1 Induzível por Hipóxia , Inflamação , MicroRNAs/genética , Óxido Nítrico Sintase Tipo II , Ratos , Choque Hemorrágico/genética , Choque Hemorrágico/terapia , Baço/fisiopatologiaRESUMO
The biomimetic pancreatic microenvironment improves the differentiation efficiency and function of human embryonic stem cell-derived ß-cells (SC-ß cells). Thus, a laminin subunit alpha 2-gelatin methacrylate (LAMA2-GelMA) hybrid hydrogel as a bionics carrier for the formation and maturation of endocrine lineage was developed in our research, based on pancreas proteomics analysis of postnatal mice. Pancreatic endocrine cells cultured on the hybrid hydrogel in vitro, which was composed of 0.5 µg/mL LAMA2 protein and 4% GelMA, the expression of transcription factors (TFs), including NKX6.1, NKX6.2, and NEUROD1 were upregulated. Single-cell transcriptomics was performed after LAMA2 knockdown during the early differentiation of pancreatic progenitor (PP) cells, a marked decrease in the forkhead box protein A2 (FOXA2+)/GATA-binding factor 6 (GATA6+) cluster was detected. Also, we clarified that as a receptor of LAMA2, integrin subunit alpha 7 (ITGA7) participated in Integrin-AKT signaling transduction and influenced the protein levels of FOXA2 and PDX1. In vivo experiments showed that, PP cells encapsulated in the LAMA2-GelMA hydrogel exhibited higher serum C-peptide levels compared to the GelMA and Matrigel groups in nude mice and reversed hyperglycemia more quickly in STZ-induced diabetic nude mice. Taken together, our findings highlighted the feasibility of constructing a pancreas-specific microenvironment based on proteomics and tissue engineering for the treatment of diabetes.
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Gelatina , Hidrogéis , Humanos , Camundongos , Animais , Hidrogéis/metabolismo , Camundongos Nus , Metacrilatos , Proteínas de Homeodomínio/genética , Diferenciação Celular/fisiologia , Pâncreas/metabolismo , Integrinas/metabolismoRESUMO
OBJECTIVE: To evaluate the effects of insulin gene transcription regulators PDX-1, NeuroD1 and MafA on the differentiation of bone marrow mesenchymal stem cells (mMSCs) into insulin-producing cells. METHODS: Murine mMSCs were isolated, cultured and expanded. The base sequences of transcription factors PDX-1, NeuroD1 and MafA were obtained by total gene synthesis and the recombinant adenovirus vectors harboring target genes constructed and transfected into packaging cell line 293A. mMSCs were infected with adenovirus separately or together, and then differentiated in vitro into insulin-producing cells. Reverse transcription-polymerase chain reaction (RT-PCR) was utilized to detect insulin gene expression, immunofluorescence for identifying the presence of insulin protein and insulin enzyme-linked immunosorbent assay (ELISA) for evaluating the secretory volume of insulin. RESULTS: The differentiation extent of mMSCs into ß-cell was analyzed. The ß-cell-specific transcriptional regulators and insulin gene were expressed in mMSCs after transfection. Immunofluorescent analyses revealed the activated expression of insulin in the cytoplasm of differentiated cells. A significant content of insulin was released in these cells in response to a certain concentrations of glucose stimulation. The insulin content of mMSCs infected with a combination of three transcription factors was significantly higher than that of the control group [(112.84 ± 9.67) mU/L vs (1.60 ± 0.22) mU/L, P < 0.05]. CONCLUSION: After modification by transcriptional factors PDX-1, NeuroD1 and MafA, mMSCs can secrete insulin through starting endogenous insulin gene transcription.
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Células da Medula Óssea/citologia , Diferenciação Celular , Células Secretoras de Insulina/citologia , Células-Tronco Mesenquimais/citologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Expressão Gênica , Vetores Genéticos , Proteínas de Homeodomínio/genética , Fatores de Transcrição Maf Maior/genética , Camundongos , Proteínas do Tecido Nervoso/genética , Organismos Geneticamente Modificados , Transativadores/genéticaRESUMO
OBJECTIVE: Exosomes have emerged as potential tools for the differentiation of induced pluripotent stem cells (iPSCs) into insulin-producing cells (IPCs). Exosomal microRNAs are receiving increasing attention in this process. Here, we aimed at investigating the role of exosomes derived from a murine pancreatic ß-cell line and identifying signature exosomal miRNAs on iPSCs differentiation. METHODS: Exosomes were isolated from MIN6 cells and identified with TEM, NTA and Western blot. PKH67 tracer and transwell assay were used to confirm exosome delivery into iPSCs. qRT-PCR was applied to detect key pancreatic transcription gene expression and exosome-derived miRNA expression. Insulin secretion was determined using FCM and immunofluorescence. The specific exosomal miRNAs were determined via RNA-interference of Ago2. The therapeutic effect of 21 day-exosome-induced IPCs was validated in T1D mice induced by STZ. RESULTS: iPSCs cultured in medium containing exosomes showed sustained higher expression of MAFA, Insulin1, Insulin2, Isl1, Neuroud1, Nkx6.1 and NGN3 compared to control iPSCs. In FCM analysis, approximately 52.7% of the differentiated cells displayed insulin expression at the middle stage. Consistent with the gene expression data, immunofluorescence assays showed that Nkx6.1 and insulin expression in iPSCs were significantly upregulated. Intriguingly, the expression of pancreatic markers and insulin was significantly decreased in iPSCs cultured with siAgo2 exosomes. Transplantation of 21 day-induced IPCs intoT1D mice efficiently enhanced glucose tolerance and partially controlled hyperglycemia. The therapeutic effect was significantly attenuated in T1D mice that received iPSCs cultured with siAgo2 exosomes. Of the seven exosomal microRNAs selected for validation, miR-706, miR-709, miR-466c-5p, and miR-423-5p showed dynamic expression during 21 days in culture. CONCLUSION: These data indicate that differentiation of exosome-induced iPSCs into functional cells is crucially dependent on the specific miRNAs encased within exosomes, whose functional analysis is likely to provide insight into novel regulatory mechanisms governing iPSCs differentiation into IPCs.
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Ophiopogonin, including Ophiopogonin A, B, C, D, is an effective active component of traditional Chinese medicine Ophiopogon japonicus which has a wide range of pharmacological effects such as protecting myocardial ischemia, resisting myocardial infarction, immune regulation, lowering blood glucose, and anti-tumor. However, the functions of ophiopogonin A on hemorrhagic shock (HS)-induced renal injury remain unclear. First, this study constructed an HS rat model and hypoxia HK-2 cell model to assess the effects of ophiopogonin A in vivo and in vitro. In vivo, HE and TUNEL staining show that ophiopogonin A dose-dependently inhibits HS-induced tissue damage and apoptosis. Moreover, ophiopogonin A dose-dependently downregulates the levels of blood urea nitrogen (BUN), creatinine (Cr), KIM-1, NGAL, iNOS, TNF-α, IL-1ß, and IL-6 in HS rats kidney tissues, and decreases the number of MPO-positive cells. In vitro, we get similar results that ophiopogonin A dose-dependently improves hypoxia-induced HK-2 cell apoptosis and damage. In addition, ophiopogonin A dose-dependently increases the expression of NF E2-related factor 2 (Nrf2), while knockdown of Nrf2 reverses the functions of ophiopogonin A in vivo and in vitro. Furthermore, ophiopogonin A dose-dependently promotes the phosphorylation of ERK in HS kidney tissues and hypoxia-treated HK-2 cells, suggesting that ophiopogonin A functions via the p-ERK/ERK signaling pathway.
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The ultimate goal of pancreatic tissue engineering is to create a long-lived substitute organ to treat diabetes. However, the lack of neovascularization and the occurrence of immune response limit the efficacy of tissue-engineered pancreas after in vivo transplantation. Platelet-rich plasma (PRP) is an autologous platelet concentrate containing a large number of growth factors and immunoregulatory factors. The aim of this study was to evaluate rat pancreatic decellularized scaffold (PDS) loaded with PRP for vascularization, host inflammatory response and macrophage polarization in an animal model. The study results indicated that compared to PDS, PRP-loading PDS exhibited the enhanced mechanical properties and released growth factors in a slow and sustained manner to supplement the loss of growth factors during decellularization. In vitro, human umbilical vein endothelial cells (HUVECs) were seeded in PDS and PRP-loading PDS, and cultured in the circular perfusion system. When compared with PDS, PRP-loading PDS significantly promoted the colonization, proliferation and pro-angiogenic genes expression of cells on scaffolds. In vivo, PDS loaded with PRP then re-endothelialized with HUVECs were implanted subcutaneously in rats, which enhanced the angiogenesis of scaffolds, inhibited the host inflammatory response, and induced the polarization dominated by pro-regenerative M2 macrophages that also facilitated tissue vascular regeneration. Thus, the re-endothelialized PRP-loading PDS may represent a promising bioengineered pancreas with sustained vascularization and excellent biocompatibility.
Assuntos
Materiais Biocompatíveis/química , Vasos Sanguíneos/química , Plasma Rico em Plaquetas/metabolismo , Alicerces Teciduais/química , Animais , Materiais Biocompatíveis/metabolismo , Vasos Sanguíneos/metabolismo , Adesão Celular , Proliferação de Células , Células Endoteliais da Veia Umbilical Humana , Humanos , Macrófagos/metabolismo , Fenômenos Mecânicos , Neovascularização Fisiológica , Pâncreas , Ratos , Engenharia TecidualRESUMO
Eighteen new ß-carboline-based indole-4,7-quinone derivatives (12a-i and 13a-i) were designed and synthesized, and their in vitro and in vivo antiproliferative activities were studied. Most of target compounds showed strong inhibition on three human tumor cells' proliferation. In particular, the most active compound 13g not only displayed more prominent antiproliferative activities than ß-lapachone, a clinical antitumor candidate, but also exerted significant NAD(P)H: quinone-oxidoreductase-1 (NQO1) inhibitory activity and NQO1-dependent cytotoxicity in HT29 cells. Furthermore, 13g dose-dependently induced high ROS levels in HT29 cells, and selectively inhibited cancer cell but not non-tumor colon cell proliferation in vitro. Importantly, 13g promoted HT29 cell apoptosis and DNA damage by regulating relative apoptotic proteins and H2AX expression. Finally, 13g displayed significant growth inhibition of HT29 human colorectal adenocarcinoma xenograft in mice without overt toxicity.