RESUMO
Insulin resistance, the hallmark of type 2 diabetes mellitus (T2DM), has emerged as a pathological feature in Alzheimer's disease (AD). Given the shared role of insulin resistance in T2DM and AD, repurposing peripheral insulin sensitizers is a promising strategy to preserve neuronal insulin sensitivity and prevent AD. 1-Deoxynojirimycin (DNJ), a bioactive iminosugar, exhibited insulin-sensitizing effects in metabolic tissues and was detected in brain tissue post-oral intake. However, its impact on brain and neuronal insulin signaling has not been described. Here, we investigated the effect of DNJ treatment on insulin signaling and AD markers in insulin-resistant human SK-N-SH neuroblastoma, a cellular model of neuronal insulin resistance. Our findings show that DNJ increased the expression of insulin signaling genes and the phosphorylation status of key molecules implicated in insulin resistance (Y1146-pIRß, S473-pAKT, S9-GSK3B) while also elevating the expression of glucose transporters Glut3 and Glut4, resulting in higher glucose uptake upon insulin stimuli. DNJ appeared to mitigate the insulin resistance-driven increase in phosphorylated tau and Aß1-42 levels by promoting insulin-induced phosphorylation of GSK3B (a major tau kinase) and enhancing mRNA expression of the insulin-degrading enzyme (IDE) pivotal for insulin and Aß clearance. Overall, our study unveils probable mechanisms underlying the potential benefits of DNJ for AD, wherein DNJ attenuates tau and amyloid pathologies by reversing neuronal insulin resistance. This provides a scientific basis for expanding the use of DNJ-containing products for neuroprotective purposes and prompts further research into compounds with similar mechanisms of action.
Assuntos
1-Desoxinojirimicina , Doença de Alzheimer , Resistência à Insulina , Neurônios , Doença de Alzheimer/metabolismo , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/patologia , Humanos , 1-Desoxinojirimicina/farmacologia , 1-Desoxinojirimicina/análogos & derivados , Neurônios/metabolismo , Neurônios/efeitos dos fármacos , Linhagem Celular Tumoral , Peptídeos beta-Amiloides/metabolismo , Proteínas tau/metabolismo , Transportador de Glucose Tipo 3/metabolismo , Transportador de Glucose Tipo 3/genética , Insulina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transportador de Glucose Tipo 4/metabolismo , Transportador de Glucose Tipo 4/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Fosforilação/efeitos dos fármacos , Biomarcadores/metabolismoRESUMO
Sheep are important domestic animals for the production of wool and meat. Although numerous cultured cell lines from humans and mice have been established, the number of cell lines derived from sheep is limited. To overcome this issue, the efficient establishment of a sheep-derived cell line and its biological characterization is reported. Mutant cyclin-dependent kinase 4, cyclin D1, and telomerase reverse transcriptase were introduced into sheep muscle-derived cells in an attempt to immortalize primary cells using the K4DT method. Furthermore, the SV40 large T oncogene was introduced into the cells. The successful immortalization of sheep muscle-derived fibroblasts was shown using the K4DT method or SV40 large T antigen. Furthermore, the expression profile of established cells showed close biological characteristics of ear-derived fibroblasts. This study provides a useful cellular resource for veterinary medicine and cell biology.
Assuntos
Telomerase , Transcriptoma , Humanos , Animais , Camundongos , Ovinos , Linhagem Celular , Ciclo Celular , Telomerase/genética , Telomerase/metabolismo , Fibroblastos/metabolismoRESUMO
The Amami rabbit (Pentagulus furnessi) is a dark brown-furred rabbit classified as an endangered species and only found in the Amami Islands of Japan. They are often called living fossils because they retain primitive characteristics of ancient rabbits that lived approximately 1 million years ago, such as short feet and hind legs and small ears. Although the ancient rabbit has disappeared due to the competition with European rabbit (Oryctolagus cuniculus) in the most of the Asian area, Amami rabbit survived since Amami Islands has isolated from Japan and Taiwan. Although Amari rabbit is one of the protected animals, their population decreases each year due to human activities, such as deforestation and roadkill. In this study, we collected roadkill samples of Amami rabbits and established primary and immortalized fibroblast cell lines. Combined expression of human-derived mutant Cyclin-dependent kinase 4, Cyclin D1, and hTERT allowed us to immortalize fibroblasts successfully in three individuals of Amami rabbits. The immortalized fibroblasts dramatically extended the cell culture period, when it was compared with the cell culture period of wild type cells. Furthermore, the immortalized cells maintained their normal chromosomal pattern (2n = 46). Our results suggest that cellular senescence which mainly regulated by p16-RB signaling pathway is conserved in animal evolution at least from 1 million years ago.
Assuntos
Pontos de Checagem do Ciclo Celular/genética , Ciclina D1/metabolismo , Quinase 4 Dependente de Ciclina/metabolismo , Telomerase/metabolismo , Animais , Bovinos , Linhagem Celular , Células Cultivadas , Senescência Celular/fisiologia , Cromossomos/genética , Cromossomos/metabolismo , Ciclina D1/genética , Quinase 4 Dependente de Ciclina/genética , Regulação da Expressão Gênica/genética , Japão , Técnicas de Transferência Nuclear , Coelhos , Transdução de Sinais/genética , Telomerase/genéticaRESUMO
Although immortalized cultured cells are useful for various functional assays or transcriptome analysis, highly efficient and reproducible immortalization methods have not been developed in avian-derived cells. We introduced the simian virus 40 T antigen (SV40T) and human papillomavirus (HPV)-E6E7 to chick and Okinawa rail (endangered species)derived fibroblast. As a result, neither the SV40T nor E6E7 genes could induce avian cell immortality. Accordingly, we attempted to use a recently developed immortalization method, which involved the coexpression of mutant cyclin-dependent kinase 4 (CDK4), Cyclin D, and TERT (K4DT method) in these avian cells. Although the K4DT method could not efficiently induce the efficient immortalization in mass cell population, cellular division until the senescence was significantly extended by K4DT, we succeeded to obtain the immortalized avian cells (chick K4DT: one clone, Okinawa rail K4DT: three clones, Okinawa rail K4DT + telomerase RNA component: one clone) with K4DT expression. We conclude that K4DT expression is used to extend the cell division and immortalization of avian-derived cells.
Assuntos
Ciclo Celular/genética , Proliferação de Células/genética , Fibroblastos/metabolismo , RNA/genética , Telomerase/genética , Animais , Ciclo Celular/fisiologia , Divisão Celular/genética , Divisão Celular/fisiologia , Linhagem Celular , Proliferação de Células/fisiologia , Células Cultivadas , Galinhas , Genes cdc/genéticaRESUMO
Many functional foods or physiologically active ingredients derived from plants and animals are actively being investigated for their role in chronic disease prevention. Squalene (SQ) is found as active ingredient in the functional foods predominantly present in olive oil and shark liver oil. It is known that during chemotherapy anticancer drugs induce inflammation. SQ has been thought to prevent and suppress inflammation; however, there is little direct evidence available. We examined the adjuvant effect of SQ on tumor-transplanted mice along with anticancer drug doxorubicin (DOX). SQ significantly suppressed the DOX-induced increase in prostaglandin E2 (PGE2) concentration (P < 0.05) in plasma of tumor-bearing mice. SQ inhibited the numbers of writhing response (P < 0.05), formalin-induced pain and decreased COX-2 and substance P expression in the tumor tissue compared to control mice and also enhanced the antitumor efficacy of DOX in allograft mice. Thus, SQ reduces inflammation through modulation of PGE2 production indicating its potential as an adjuvant during chemotherapy in tumor-bearing mice.
Assuntos
Analgésicos/farmacologia , Anti-Inflamatórios não Esteroides/farmacologia , Alimento Funcional , Esqualeno/farmacologia , Aloenxertos , Ração Animal , Animais , Linhagem Celular Tumoral , Neoplasias do Colo/tratamento farmacológico , Ciclo-Oxigenase 2/metabolismo , Inibidores de Ciclo-Oxigenase 2/farmacologia , Dinoprostona/sangue , Doxorrubicina/administração & dosagem , Camundongos Endogâmicos BALB C , Esqualeno/administração & dosagem , Substância P/metabolismoRESUMO
Telomerase is expressed in ~90% of human cancer cell lines and tumor specimens, whereas its enzymatic activity is not detectable in most human somatic cells, suggesting that telomerase represents a highly attractive target for selective cancer treatment. Accordingly, various classes of telomerase inhibitors have been screened and developed in recent years. We and other researchers have successfully found that some dietary compounds can modulate telomerase activity in cancer cells. Telomerase inhibitors derived from food are subdivided into two groups: one group directly blocks the enzymatic activity of telomerase (e.g., catechin and sulfoquinovosyldiacylglycerol), and the other downregulates the expression of human telomerase reverse transcriptase (hTERT), the catalytic subunit of human telomerase, via signal transduction pathways (e.g., retinoic acid and tocotrienol). In contrast, a few dietary components, including genistein and glycated lipid, induce cellular telomerase activity in several types of cancer cells, suggesting that they may be involved in tumor progression. This review summarizes the current knowledge about the effects of dietary factors on telomerase regulation in cancer cells and discusses their molecular mechanisms of action.
Assuntos
Dieta , Neoplasias/genética , Neoplasias/metabolismo , Telomerase/metabolismo , Animais , Proteínas de Ligação a DNA/metabolismo , Suplementos Nutricionais , Ativação Enzimática/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Regiões Promotoras Genéticas , Ligação Proteica , Telomerase/genética , Transcrição GênicaRESUMO
In this study, we created porcine-induced pluripotent stem (iPS) cells with the expression of six reprogramming factors (Oct3/4, Klf4, Sox2, c-Myc, Lin28, and Nanog). The resulting cells showed growth dependent on LIF (leukemia inhibitory factor) and expression of multiple stem cell markers. Furthermore, the iPS cells caused teratoma formation with three layers of differentiation and had both active X chromosomes (XaXa). Our iPS cells satisfied the both of important characteristics of stem cells: teratoma formation and activation of both X chromosomes. Injection of these iPS cells into morula stage embryos showed that these cells participate in the early stage of porcine embryogenesis. Furthermore, the RNA-Seq analysis detected that expression levels of endogenous pluripotent related genes, NANOG, SOX2, ZFP42, OCT3/4, ESRRB, and ERAS were much higher in iPS with six factors than that with four reprogramming factors. We can conclude that the expression of six reprogramming factors enables the creation of porcine iPS cells, which is partially close to naive iPS state. J. Cell. Biochem. 118: 537-553, 2017. © 2016 Wiley Periodicals, Inc.
Assuntos
Técnicas de Reprogramação Celular , Expressão Gênica , Células-Tronco Pluripotentes Induzidas/metabolismo , Fatores de Transcrição/biossíntese , Cromossomo X/metabolismo , Animais , Feminino , SuínosRESUMO
Tocotrienol (T3), unsaturated vitamin E, is gaining a lot of attention owing to its potent anticancer effect, since its efficacy is much greater than that of tocopherol (Toc). Various factors are known to be involved in such antitumor action, including cell cycle arrest, apoptosis induction, antiangiogenesis, anti-metastasis, nuclear factor-κB suppression, and telomerase inhibition. Owing to a difference in the affinity of T3 and Toc for the α-tocopherol transfer protein, the bioavailability of orally ingested T3 is lower than that of Toc. Furthermore, cellular uptake of T3 is interrupted by coadministration of α-Toc in vitro and in vivo. Based on this, several studies are in progress to screen for molecules that can synergize with T3 in order to augment its potency. Combinations of T3 with chemotherapeutic drugs (e.g., statins, celecoxib, and gefitinib) or dietary components (e.g., polyphenols, sesamin, and ferulic acid) exhibit synergistic actions on cancer cell growth and signaling pathways. In this review, we summarize the current status of synergistic effects of T3 and an array of agents on cancer cells, and discuss their molecular mechanisms of action. These combination strategies would encourage further investigation and application in cancer prevention and therapy.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias/tratamento farmacológico , Tocotrienóis/uso terapêutico , Catequina/análogos & derivados , Catequina/uso terapêutico , Ácidos Cumáricos/uso terapêutico , Suplementos Nutricionais , Dioxóis/uso terapêutico , Sinergismo Farmacológico , Quimioterapia Combinada , Humanos , Lignanas/uso terapêutico , Neoplasias/prevenção & controle , Resveratrol , Estilbenos/uso terapêuticoRESUMO
Nonhuman primates are useful animal models for the study of human diseases. However, the number of established cell lines from nonhuman primates is quite limited compared with the number established from other experimental animals. The establishment of nonhuman primate cell lines would allow drug testing on those cell lines before moving experiments into primates. In this study, we established nonhuman primate primary cell lines by introducing the genes for CDK4R24C, cyclin D1, and hTERT. These cell lines proliferated more rapidly than primary cells and bypassed cellular senescence. Karyotype analysis showed that the chromosome patterns were intact in the immortalized cell lines. Furthermore, we showed that the expression of introduced genes could be precisely controlled through the Tet-Off system with the addition of doxycycline. The present study shows that introduction of the CDK4R24C, cyclin D1, and hTERT genes are effective methods of establishing nonhuman primate cell lines.
Assuntos
Proteínas de Ciclo Celular/genética , Fibroblastos/citologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica/genética , Animais , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Linhagem Celular Transformada , Proliferação de Células/genética , Células Cultivadas , Senescência Celular/genética , Ciclina D1/genética , Ciclina D1/metabolismo , Quinase 4 Dependente de Ciclina/genética , Quinase 4 Dependente de Ciclina/metabolismo , Doxiciclina/farmacologia , Feminino , Fibroblastos/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Vetores Genéticos/genética , Humanos , Immunoblotting , Cariótipo , Lentivirus/genética , Macaca , Masculino , Mutação de Sentido Incorreto , Telomerase/genética , Telomerase/metabolismo , TransfecçãoRESUMO
In this study, we investigated cellular uptake and metabolism of phosphatidylcholine hydroperoxide (PCOOH) in human hepatoma HepG2 cells by high performance liquid chromatography-tandem mass spectrometry, and then evaluated whether PCOOH or its metabolites cause pathophysiological effects such as cytotoxicity and apoptosis. Although we found that most PCOOH was reduced to PC hydroxide in HepG2 cells, the remaining PCOOH caused cytotoxic effects that may be mediated through an unusual apoptosis pathway. These results will enhance our fundamental understanding of how PCOOH, which is present in oxidized low density lipoproteins, is involved in the development of atherosclerosis.
Assuntos
Apoptose , Células Hep G2/citologia , Fosfatidilcolinas/metabolismo , Ciclo Celular , Células Hep G2/metabolismo , Células Hep G2/patologia , Humanos , Potencial da Membrana Mitocondrial , Fosfatidilcolinas/toxicidadeRESUMO
Rice bran consists of many functional compounds and thus much attention has been focused on the health benefits of its components. Here, we investigated the synergistic inhibitory effects of its components, particularly δ-tocotrienol (δ-T3) and ferulic acid (FA), against the proliferation of an array of cancer cells, including DU-145 (prostate cancer), MCF-7 (breast cancer), and PANC-1 (pancreatic cancer) cells. The combination of δ-T3 and FA markedly reduced cell proliferation relative to δ-T3 alone, and FA had no effect when used alone. Although δ-T3 induced G1 arrest by up-regulating p21 in PANC-1 cells, more cells accumulated in G1 phase with the combination of δ-T3 and FA. This synergistic effect was attributed to an increase in the cellular concentration of δ-T3 by FA. Our results suggest that the combination of δ-T3 and FA may present a new strategy for cancer prevention and therapy.
Assuntos
Proliferação de Células/efeitos dos fármacos , Ácidos Cumáricos/farmacologia , Neoplasias/patologia , Vitamina E/análogos & derivados , Linhagem Celular Tumoral , Sinergismo Farmacológico , Humanos , Reação em Cadeia da Polimerase em Tempo Real , Vitamina E/farmacologiaRESUMO
The Ryukyu long-furred rat is an endangered species confined to the southernmost three small islands of Japan (Amami-Oshima, Tokunoshima, and Okinawa). Its population is rapidly decreasing because of roadkill, deforestation, and feral animals. To date, its genomic and biological information are poorly understood. In this study, we successfully immortalized Ryukyu long-furred rat cells by expressing a combination of cell cycle regulators, mutant cyclin-dependent kinase 4 (CDK4R24C) and cyclin D1, together with telomerase reverse transcriptase or an oncogenic protein, the Simian Virus large T antigen. The cell cycle distribution, telomerase enzymatic activity, and karyotype of these two immortalized cell lines were analyzed. The karyotype of the former cell line immortalized with cell cycle regulators and telomerase reverse transcriptase retained the nature of the primary cells, while that of the latter cell line immortalized with the Simian Virus large T antigen had many aberrant chromosomes. These immortalized cells would be valuable for studying the genomics and biology of Ryukyu long-furred rats.
Assuntos
Telomerase , Ratos , Animais , Telomerase/genética , Telomerase/metabolismo , Divisão Celular , Ciclo Celular , Linhagem Celular , Antígenos Virais de Tumores/genéticaRESUMO
Vitamin E is classified into tocopherol (Toc) and tocotrienol (T3) based on its side chains. T3 generally has higher cellular uptake than Toc, though the responsible mechanism remains unclear. To elucidate this mechanism, we hypothesized and investigated whether serum albumin is a factor that induces such a difference in the cellular uptake of Toc and T3. Adding bovine serum albumin (BSA) to serum-depleted media increased the cellular uptake of T3 and decreased that of Toc, with varying degrees among α-, ß-, γ-, and δ-analogs. Such enhanced uptake of α-T3 was not observed when cells were incubated under low temperature (the uptake of α-Toc was also reduced), suggesting that Toc and T3 bind to albumin to form a complex that results in differential cellular uptake of vitamin E. Fluorescence quenching study confirmed that vitamin E certainly bound to BSA, and that T3 showed a higher affinity than Toc. Molecular docking further indicated that the differential binding energy of Toc or T3 to BSA is due to the Van der Waals interactions via their side chain. Overall, these results suggested that the affinity of Toc and T3 to albumin differs due to their side chains, causing the difference in their albumin-mediated cellular uptake. Our results give a better mechanistic insight into the physiological action of vitamin E.
Assuntos
Tocoferóis , Tocotrienóis , Tocoferóis/farmacologia , Simulação de Acoplamento Molecular , Vitamina E/metabolismo , Albuminas , Soroalbumina BovinaRESUMO
Previously, we reported that near-infrared irradiation that simulates solar near-infrared irradiation with pre- and parallel-irradiational cooling can non-thermally induce cytocidal effects in cancer cells. To explore these effects, we assessed cell viability, DNA damage response pathways, and the percentage of mitotic cancer cells after near-infrared treatment. Further, we evaluated the anti-cancer effects of near-infrared irradiation compared with doxorubicin in xenografts in nude mice by measuring tumor volume and assessing protein phosphorylation by immunoblot analysis. The cell viability of A549 lung adenocarcinoma cells was significantly decreased after three rounds of near-infrared irradiation at 20 J/cm(2). Apoptotic cells were observed in near-infrared treated cells. Moreover, near-infrared treatment increased the phosphorylation of ataxia-telangiectasia mutated (ATM) at Ser(1981), H2AX at Ser(139), Chk1 at Ser(317), structural maintenance of chromosome (SMC) 1 at Ser(966), and p53 at Ser(15) in A549 cells compared with control. Notably, near-infrared treatment induced the formation of nucleic foci of γH2AX. The percentage of mitotic A549 cells, as measured by histone H3 phosphorylation, decreased significantly after three rounds of near-infrared irradiation at 20 J/cm(2). Both near-infrared and doxorubicin inhibited the tumor growth of MDA-MB435 melanoma cell xenografts in nude mice and increased the phosphorylation of p53 at Ser(15), Chk1 at Ser(317), SMC1 at Ser(966), and H2AX at Ser(139) compared with control mice. These results indicate that near-infrared irradiation can non-thermally induce cytocidal effects in cancer cells as a result of activation of the DNA damage response pathway. The near-infrared irradiation schedule used here reduces discomfort and side effects. Therefore, this strategy may have potential application in the treatment of cancer.
Assuntos
Adenocarcinoma/genética , Apoptose/efeitos da radiação , Sobrevivência Celular/efeitos da radiação , Dano ao DNA , Neoplasias Pulmonares/genética , Adenocarcinoma de Pulmão , Animais , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Feminino , Pontos de Checagem da Fase G2 do Ciclo Celular , Humanos , Immunoblotting , Camundongos , Camundongos Nus , Índice MitóticoRESUMO
The pig is an important animal for both agricultural and medical purposes. However, the number of pig-derived cell lines is relatively limited when compared with mouse- and human-derived lines. We established in this study a retroviral conditional expression system for the Simian vacuolating virus 40 large T fragment (SV40T) which allowed us to efficiently establish pig embryonic fibroblast cell lines. The established cell lines showed high levels of cell proliferation and resistance to cellular senescence. A chromosome analysis showed that 84% of the cells had the normal karyotype. Transient expression of the Cre recombinase allowed us to excise the SV40T fragment from the genome. The development of this research tool will enable us to quickly establish new cell lines derived from various animals.
Assuntos
Antígenos Transformantes de Poliomavirus/genética , Linhagem Celular , Fibroblastos/citologia , Vírus 40 dos Símios/genética , Animais , Proliferação de Células , Embrião de Mamíferos , Fibroblastos/metabolismo , Fibroblastos/virologia , Efeito Fundador , Expressão Gênica , Engenharia Genética , Integrases/genética , Cariótipo , Cariotipagem , SuínosRESUMO
Common marmosets are non-human primate models used in biomedical research and genome editing technology. This study aimed to establish cell lines from common marmosets and evaluate their characteristics. We obtained normal fibroblasts derived from muscle tissues of two common marmosets and immortalized them with the introduction of a mutat form of cyclin-dependent kinase 4 (CDK4R24C), Cyclin D1, and telomere reverse transcriptase (TERT) using the piggyBac transposon. Compared to parental cells, the immortalized cell lines (named K4DT cells) showed telomerase activity and an accelerated cell proliferation rate. To our knowledge, this is the first study describing the successful establishment of immortalized common marmoset-derived fibroblasts using piggyBac transposition of CDK4R24C, Cyclin D1, and TERT. Our generated cell lines might be a beneficial tool for future studies on disease modeling and targeted gene therapies.
Assuntos
Callithrix , Telomerase , Animais , Callithrix/metabolismo , Ciclo Celular/genética , Linhagem Celular , Ciclina D1/metabolismo , Fibroblastos/metabolismo , Telomerase/genética , Telomerase/metabolismoRESUMO
Ethanolamine plasmalogen (PlsEtn) is a subtype of ethanolamine glycerophospholipids (EtnGpl). Recently, PlsEtn has attracted increasing research interest due to its beneficial effects in health and disease; however, its functional role in colonic health has not been well established. This study was conducted to determine the mechanism underlying the antiapoptotic effect of PlsEtn in human intestinal tract cells under induced inflammatory stress. Lipopolysaccharide induced apoptosis of differentiated Caco-2 cells, which was suppressed by EtnGpl in a dose-dependent manner. Cells treated with ascidian muscle EtnGpl containing high levels of PlsEtn demonstrated a lower degree of apoptosis, and downregulated TNF-α and apoptosis-related proteins compared to those treated with porcine liver EtnGpl containing low PlsEtn. This indicates that PlsEtn exerted the observed effects, which provided protection against induced inflammatory stress. Overall, our results suggest that PlsEtn with abundant vinyl ether linkages is potentially beneficial in preventing the initiation of inflammatory bowel disease and colon cancer.
RESUMO
Clinical studies have recently demonstrated that autologous transplantation of mobilized dental pulp stem cells is a safe and efficacious potential therapy for pulp regeneration. However, some limitations need to be addressed, such as the high cost of the safety and quality control tests for isolated individual dental pulp cell products before transplantation. Therefore, more efficient in vitro culturing of human dental pulp stem cells might be useful for providing low cost and high reliability testing for pulp regeneration therapy. In this study, we established a novel immortalized dental pulp stem cell line by co-expressing a mutant cyclin-dependent kinase 4 (CDK4R24C), Cyclin D1, and telomerase reverse transcriptase (TERT). The established cell line maintained its original diploid chromosomes and stemness characteristics and exhibited an enhanced proliferation rate. In addition, we showed the immortalized human dental pulp stem cells still keeps their osteogenic and adipogenic differentiation abilities under appropriate culture conditions even though the cell proliferation was accelerated. Taken together, our established cell lines could serve as a useful in vitro tool for pulp regeneration therapy, and can contribute to reproducibility and ease of cell handling, thereby saving time and costs associated with safety and quality control tests.
Assuntos
Ciclo Celular/genética , Cromossomos Humanos/genética , Polpa Dentária/citologia , Transplante de Células-Tronco , Células-Tronco/citologia , Proliferação de Células , Senescência Celular , Ciclina D1/genética , Quinase 4 Dependente de Ciclina/genética , Expressão Gênica , Humanos , Telomerase/genéticaRESUMO
Androgenetic alopecia (AGA) is the most common type of hair loss, and is mainly caused by the biological effects of testosterone on dermal papilla cells (DPCs). In vitro culturing of DPCs might be a useful tool for the screening of target molecule of AGA. However, primary DPCs cannot continuously proliferate owing to cellular senescence and cell culture stress. In this study, we introduced mutant cyclin-dependent kinase 4 (CDK4), Cyclin D1, and telomerase reverse transcriptase (TERT) into DPCs. We confirmed protein expression of CDK4 and Cyclin D1, and enzymatic activity of TERT. Furthermore, we found the established cell line was free from cellular senescence. We also introduced the androgen receptor gene using a recombinant retrovirus, to compensate the transcriptional suppressed endogenous androgen receptor in the process of cell proliferation. Furthermore, we detected the efficient nuclear translocation of androgen receptor into the nucleus after the treatment of dihydrotestosterone, indicating the functionality of our introduced receptor. Our established cell line is a useful tool to identify the downstream signaling pathway, which activated by the testosterone.
RESUMO
1-Deoxynojirimycin (DNJ) has been known for its functional properties, such as its anti-hyperglycemic and anti-obesity activities. Previously, we developed a sustainable procedure to produce culture broth powder (CBP) containing DNJ using Bacillus amyloliquefaciens AS385 and demonstrated its regulatory effect on the blood glucose and lipid parameters in C57BL/6J mice. The present study was aimed to determine the molecular mechanism underlying the physiological effects of CBP intake in different concentrations (low, medium and high) towards the development of high-fat diet (HFD)-induced metabolic disorders. Ten-week consumption of CBP-supplemented diets ameliorated HFD-induced adiposity, glucose intolerance, and reduced insulin sensitivity in C57BL/6J mice. To investigate how these physiological events could take place, we analyzed the expression of genes involved in lipid metabolism and insulin signaling in epididymal white adipose tissue and found that CBP had a regulatory effect on the expression of genes related to lipid metabolism (Pparγ, Srebf1c, Acc, Scd, Hsl, Lpl), adiponectin secretion (Foxo1 and Sirt1), and insulin signaling (Irs1 and Akt2). Next, we confirmed that DNJ acted as the main active component in CBP and detected the dose-dependent DNJ uptake in vital metabolic tissues, which may explain the dose-dependent alteration in the metabolic parameters and related gene expressions following the CBP intake in this study. Collectively, our results suggested that DNJ intake in the form of CBP prevented the progression of HFD-induced metabolic disorders through regulation of adipocyte gene expression involved in lipid metabolism and insulin signaling.