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1.
Int J Mol Sci ; 23(23)2022 Nov 25.
Artículo en Inglés | MEDLINE | ID: mdl-36499074

RESUMEN

In vitro expansion-mediated replicative senescence has severely limited the clinical applications of mesenchymal stem cells (MSCs). Accumulating studies manifested that nicotinamide adenine dinucleotide (NAD+) depletion is closely related to stem cell senescence and mitochondrial metabolism disorder. Promoting NAD+ level is considered as an effective way to delay aging. Previously, we have confirmed that nicotinamide mononucleotide (NMN), a precursor of NAD+, can alleviate NAD+ deficiency-induced MSC senescence. However, whether NMN can attenuate MSC senescence and its underlying mechanisms are still incompletely clear. The present study herein showed that late passage (LP) MSCs displayed lower NAD+ content, reduced Sirt3 expression and mitochondrial dysfunction. NMN supplementation leads to significant increase in intracellular NAD+ level, NAD+/ NADH ratio, Sirt3 expression, as well as ameliorated mitochondrial function and rescued senescent MSCs. Additionally, Sirt3 over-expression relieved mitochondrial dysfunction, and retrieved senescence-associated phenotypic features in LP MSCs. Conversely, inhibition of Sirt3 activity via a selective Sirt3 inhibitor 3-TYP in early passage (EP) MSCs resulted in aggravated cellular senescence and abnormal mitochondrial function. Furthermore, NMN administration also improves 3-TYP-induced disordered mitochondrial function and cellular senescence in EP MSCs. Collectively, NMN replenishment alleviates mitochondrial dysfunction and rescues MSC senescence through mediating NAD+/Sirt3 pathway, possibly providing a novel mechanism for MSC senescence and a promising strategy for anti-aging pharmaceuticals.


Asunto(s)
Células Madre Mesenquimatosas , Enfermedades Mitocondriales , Sirtuina 3 , Senescencia Celular , Células Madre Mesenquimatosas/metabolismo , Mitocondrias/metabolismo , Enfermedades Mitocondriales/metabolismo , NAD/metabolismo , Mononucleótido de Nicotinamida/farmacología , Mononucleótido de Nicotinamida/metabolismo , Sirtuina 3/metabolismo , Animales
2.
Exp Cell Res ; 352(1): 45-52, 2017 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-28159473

RESUMEN

Decreased bone volume and strength with aging and enhanced risk of fractures are in part due to reduced number of bone-forming mesenchymal stem cells (MSCs) and cellular dysfunction. In a previous study, we found that osteogenic differentiation of the multipotent and omnipotent preosteoblasts are accompanied by the alterations of intracellular NAD metabolism in which nicotinamide phosphoribosyltransferase (Nampt) plays a regulatory role. The increased Nampt during osteoblast differentiation, the enzyme catalyzing NAD resynthesis from nicotinamide was noted. However, whether Nampt will also be able to affect osteogenic differentiation of primary bone marrow-derived mesenchymal stem cells (BM-MSCs), it is still uncertain. Here we report the role of Nampt in regulating osteoblast differentiation in primary mouse BM-MSCs. We found that Nampt expression was progressively elevated during BM-MSCs osteogenic differentiation. The Nampt inhibitor FK866 or knock-down of Nampt in BM-MSCs led to declined osteoblastogenesis, including attenuated ALP activity, diminished matrix mineralization and down-regulated osteoblast specific marker genes. In addition, declined osteoblastogenesis by Nampt deficiency or addition of FK866 was related to lower intracellular NAD concentration and decreased Sirt1 activity. The present findings demonstrate that osteogenic differentiation in MSCs can be modulated by intracellular NAD metabolism, in which Nampt may serve as an applicable marker for the osteoblast determination.


Asunto(s)
Médula Ósea/metabolismo , Diferenciación Celular , Citocinas/metabolismo , Células Madre Mesenquimatosas/citología , Nicotinamida Fosforribosiltransferasa/metabolismo , Osteoblastos/citología , Animales , Apoptosis , Western Blotting , Proliferación Celular , Células Cultivadas , Citocinas/genética , Humanos , Masculino , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , NAD/metabolismo , Nicotinamida Fosforribosiltransferasa/genética , Osteoblastos/metabolismo , Osteogénesis , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sirtuina 1/genética , Sirtuina 1/metabolismo
3.
Stem Cells ; 31(9): 1840-56, 2013 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-23666768

RESUMEN

We sought to biologically characterize and identify a subpopulation of urine-derived stem cells (USCs) with the capacity for multipotent differentiation. We demonstrated that single USCs can expand to a large population with 60-70 population doublings. Nine of 15 individual USC clones expressed detectable levels of telomerase and have long telomeres. These cells expressed pericyte and mesenchymal stem cell markers. Upon induction with appropriate media in vitro, USCs differentiated into bladder-associated cell types, including functional urothelial and smooth muscle cell lineages. When the differentiated USCs were seeded onto a scaffold and subcutaneously implanted into nude mice, multilayered tissue-like structures formed consisting of urothelium and smooth muscle. Additionally, USCs were able to differentiate into endothelial, osteogenic, chondrogenic, adipogenic, skeletal myogenic, and neurogenic lineages but did not form teratomas during the 1-month study despite telomerase activity. USCs may be useful in cell-based therapies and tissue engineering applications, including urogenital reconstruction.


Asunto(s)
Diferenciación Celular , Células Madre Multipotentes/citología , Células Madre Multipotentes/trasplante , Trasplante de Células Madre , Orina/citología , Urología , Adolescente , Adulto , Anciano , Animales , Biomarcadores/metabolismo , Linaje de la Célula , Separación Celular , Niño , Preescolar , Células Clonales , Femenino , Citometría de Flujo , Humanos , Riñón/citología , Ratones , Ratones Desnudos , Persona de Mediana Edad , Telomerasa/metabolismo , Adulto Joven
4.
Front Pharmacol ; 14: 1122065, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-36969848

RESUMEN

Renal cell carcinoma (RCC) is the most frequent renal malignancy in the world, and its incidence is increasing year by year. RCC is a well-known drug resistant tumor, and the treatment methods are limited. Most patients with RCC are discovered at the advanced stage, and thus have poor prognosis even after treatment. Therefore, it is very urgent to find new markers for the diagnosis and treatment of RCC. Accumulating evidence shows that lncRNAs participate in the occurrence and progression of RCC, which is achieved by the lncRNA-miRNA-mRNA axis. It is widely known that metabolic defect is an essential pathogenesis in RCC. As is the case with other tumors, RCC can satisfy the demands of cancerous cells for uncontrolled proliferation through aerobic glycolysis. However, whether lncRNAs can modulate RCC progression through metabolic pathway is still not clarified. Taken together, this review mainly summarized the metabolic regulatory mechanisms of lncRNAs in RCC progression, especially their roles in glucose metabolism, lipid metabolism, amino acid metabolism and mitochondrial dynamics, as well as the clinical applications of lncRNAs via targeting metabolism in RCC therapy. It will provide the new targets and approaches for early clinical diagnosis, treatment and prognosis of RCC.

5.
Ageing Res Rev ; 87: 101917, 2023 06.
Artículo en Inglés | MEDLINE | ID: mdl-36972842

RESUMEN

The skin is the largest organ of the human body and the first line of defense against environmental hazards. Many factors, including internal factors such as natural aging and external factors such as ultraviolet radiation and air pollution, can lead to skin aging. Mitochondria provide sufficient energy to maintain the high-speed turnover capacity of the skin, so the quality control of mitochondria plays an indispensable role in this process. Mitochondrial dynamics, mitochondrial biogenesis and mitophagy are the key steps in mitochondrial quality surveillance. They are coordinated to maintain mitochondrial homeostasis and restore damaged mitochondrial function. All of the mitochondrial quality control processes are related to skin aging caused by various factors. Therefore, fine-tuning regulation of the above process is of great significance to the skin aging problem that needs to be solved urgently. This article mainly reviews the physiological and environmental factors causing skin aging, the effects of mitochondrial dynamics, mitochondrial biogenesis and mitophagy on skin aging, as well as their specific regulatory mechanisms. Finally, mitochondrial biomarkers for diagnosis of skin aging, and therapeutic approaches of skin aging via mitochondrial quality control were illustrated.


Asunto(s)
Mitofagia , Envejecimiento de la Piel , Humanos , Dinámicas Mitocondriales , Rayos Ultravioleta , Mitocondrias
6.
Front Cell Dev Biol ; 10: 890574, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35693947

RESUMEN

Telomerase activity is essential for the self-renewal and potential of embryonic, induced pluripotent, and cancer stem cells, as well as a few somatic stem cells, such as human urine-derived stem cells (USCs). However, it remains unclear how telomerase activity affects the regeneration potential of somatic stem cells. The objective of this study was to determine the regenerative significance of telomerase activity, particularly to retain cell surface marker expression, multipotent differentiation capability, chromosomal stability, and in vivo tumorigenic transformation, in each clonal population of human primary USCs. In total, 117 USC specimens from 10 healthy male adults (25-57 years of age) were obtained. Polymerase chain reaction amplification of a telomeric repeat was used to detect USCs with positive telomerase activity (USCsTA+). A total of 80 USCsTA+ (70.2%) were identified from 117 USC clones, but they were not detected in the paired normal bladder smooth muscle cell and bone marrow stromal cell specimens. In the 20-40 years age group, approximately 75% of USC clones displayed positive telomerase activity, whereas in the 50 years age group, 59.2% of the USC clones expressed positive telomerase activity. USCsTA+ extended to passage 16, underwent 62.0 ± 4.8 population doublings, produced more cells, and were superior for osteogenic, myogenic, and uroepithelial differentiation compared to USCsTA-. Importantly, USCs displayed normal chromosome and no oncological transformation after being implanted in vivo. Overall, as a safe cell source, telomerase-positive USCs have a robust regenerative potential in cell proliferation and multipotent differentiation capacity.

7.
Stem Cell Rev Rep ; 18(7): 2315-2327, 2022 10.
Artículo en Inglés | MEDLINE | ID: mdl-35460064

RESUMEN

Cellular senescence is an irreversible cell arrest process, which is determined by a variety of complicated mechanisms, including telomere attrition, mitochondrial dysfunction, metabolic disorders, loss of protein homeostasis, epigenetic changes, etc. Cellular senescence is causally related to the occurrence and development of age-related disease. The elderly is liable to suffer from disorders such as neurodegenerative diseases, cancer, and diabetes. Therefore, it is increasingly imperative to explore specific countermeasures for the treatment of age-related diseases. Numerous studies on humans and mice emphasize the significance of metabolic imbalance caused by short telomeres and mitochondrial damages in the onset of age-related diseases. Although the experimental data are relatively independent, more and more evidences have shown that there is mutual crosstalk between telomeres and mitochondrial metabolism in the process of cellular senescence. This review systematically discusses the relationship between telomere length, mitochondrial metabolic disorder, as well as their underlying mechanisms for cellular senescence and age-related diseases. Future studies on telomere and mitochondrial metabolism may shed light on potential therapeutic strategies for age-related diseases. Graphical Abstract The characteristics of cellular senescence mainly include mitochondrial dysfunction and telomere attrition. Mitochondrial dysfunction will cause mitochondrial metabolic disorders, including decreased ATP production, increased ROS production, as well as enhanced cellular apoptosis. While oxidative stress reaction to produce ROS, leads to DNA damage, and eventually influences telomere length. Under the stimulation of oxidative stress, telomerase catalytic subunit TERT mainly plays an inhibitory role on oxidative stress, reduces the production of ROS and protects telomere function. Concurrently, mitochondrial dysfunction and telomere attrition eventually induce a range of age-related diseases, such as T2DM, osteoporosis, AD, etc. :increase; :reduce;⟝:inhibition.


Asunto(s)
Telomerasa , Adenosina Trifosfato/metabolismo , Anciano , Envejecimiento/genética , Animales , Senescencia Celular/genética , Humanos , Ratones , Mitocondrias/genética , Mitocondrias/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Telomerasa/metabolismo , Telómero/genética , Telómero/metabolismo
8.
Aging (Albany NY) ; 14(2): 1014-1032, 2022 01 30.
Artículo en Inglés | MEDLINE | ID: mdl-35122680

RESUMEN

The functions of stem cells decline progressively with aging, and some metabolic changes occur during the process. However, the molecular mechanisms of stem cell aging remain unclear. In this study, the combined application of metabolomics and transcriptomics technologies can effectively describe the possible molecular mechanisms of rat bone marrow mesenchymal stem cell (BMSC) senescence. Metabolomic profiles revealed 23 differential metabolites which were abundant in "glycerophospholipid metabolism", "linoleic acid metabolism" and "biosynthesis of unsaturated fatty acids". In addition, transcriptomics analysis identified 590 genes with enormously differential expressions in young and old BMSCs. KEGG enrichment analyses showed that metabolism-related pathways in BMSC senescence had stronger responses. Furthermore, the integrated analysis of the interactions between the differentially expressed genes (DEGs) and metabolites indicated the differential genes related to lipid metabolism of Scd, Scd2, Dgat2, Fads2, Lpin1, Gpat3, Acaa2, Lpcat3, Pcyt2 and Pla2g4a may be closely associated with the aging of BMSCs. Finally, Scd2 was identified as the most significant DEG, and Scd2 over-expression could alleviate cellular senescence in aged BMSCs. In conclusion, this work provides a validated understanding that the DEGs and metabolites related to lipid metabolism present more apparent changes in the senescence of rat BMSCs.


Asunto(s)
Células Madre Mesenquimatosas , Transcriptoma , Animales , Células de la Médula Ósea/metabolismo , Senescencia Celular/genética , Perfilación de la Expresión Génica , Células Madre Mesenquimatosas/metabolismo , Metabolómica , Ratas
9.
Cell Biosci ; 12(1): 62, 2022 May 14.
Artículo en Inglés | MEDLINE | ID: mdl-35568915

RESUMEN

BACKGROUND: Stem cell senescence is considered as a significant driver of organismal aging. As individuals age, the number of stem cells is declined, and the ability to proliferate and survive is also weakened. It has been reported that metabolism plays an important role in stem cell self-renewal, multilineage differentiation, senescence and fate determination, which has aroused widespread concerns. However, whether metabolism-related genes or signalling pathways are involved in physiological aging remain largely undetermined. RESULTS: In the current study, we showed 868 up-regulated and 2006 down-regulated differentially expressed genes (DEGs) in bone marrow mesenchymal stem cells (MSCs) from old rats in comparison with that from young rats by performing RNA sequence. And DEGs functions and pathways were further selected by function enrichment analysis. The results indicated that the high expression of DEGs might participate in cell differentiation, growth factor binding and etc., while the down-regulated DEGs were majorly enriched in metabolism process, such as the cellular metabolic process and mitochondria. Then, we screened and verified DEGs related to glucose metabolism and investigated the glycolysis levels. We identified that glucose uptake, lactate secretion, ATP production and relative extracellular acidification rates (ECAR) were all diminished in MSCs from old rats. More importantly, we conducted microRNA prediction on the key DEGs of glycolysis to elucidate the potential molecular mechanisms of glucose metabolism affecting MSC senescence. CONCLUSIONS: Our study unravelled the profiles of DEGs in age-associated MSC senescence and their functions and pathways. We also clarified DEGs related to glucose metabolism and down-regulated glycolysis level in age-associated MSC senescence. This study will uncover the metabolic effects on regulating stem cell senescence, and provide novel therapeutic targets for ameliorating age-associated phenotypes.

10.
Stem Cell Rev Rep ; 18(5): 1728-1742, 2022 06.
Artículo en Inglés | MEDLINE | ID: mdl-35258787

RESUMEN

Stem cell senescence and exhaustion are closely related to organ failure and individual aging, which not only induces age-related diseases, but also hinders stem cell applications in regenerative medicine. Thus, it's imminent to find effective ways to delay and retrieve stem cell senescence. Metabolic abnormalities are one of the main characteristics of age-associated declines in stem cell function. Understanding the underlying mechanisms may reveal potential strategies for ameliorating age-associated phenotypes and treating age-related diseases. This review focuses on recent advances in the association between metabolism including glucose, lipid, glutamine and NAD+ metabolism and stem cell senescence, as well as the other properties like proliferation and differentiation. Layers of studies are summarized to demonstrate how metabolism varies in senescent stem cells and how metabolic reprogramming regulates stem cell senescence. Additionally, we mentioned some recent progress in therapeutic strategies to rejuvenate dysfunctional aged stem cells. Finally, a brief conclusion about the prospect of metabolic regulation as a potential strategy for rescuing stem cell senescence is displayed. Stem cell senescence is induced by the metabolic reprogramming. The metabolic alterations of glucose, lipid, glutamine and NAD+ can conversely facilitate or inhibit stem cell senescence. Glycolysis, OXPHOS and PPP are all attenuated. But gluconeogenesis alterations still remain unclear. In lipid metabolisms, both FAO and DNL are suppressed. As for the glutamine metabolism, stem cells' dependence on glutamine is enhanced. Last, NAD+ metabolism undergoes a down-regulated synthesis and up-regulated consumption. All these alterations can be potential targets for reversing stem cell senescence.


Asunto(s)
Glutamina , NAD , Senescencia Celular/fisiología , Glucosa/metabolismo , Glutamina/farmacología , Lípidos/farmacología , NAD/metabolismo , NAD/farmacología , Células Madre
11.
Oncol Rep ; 46(6)2021 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-34608505

RESUMEN

Following the publication of this paper, an interested reader drew to the attention of the Office that the GAPDH control bands shown in Fig. 5 were strikingly similar to data appearing in different form in other articles by different authors. Owing to the fact that the contentious data in the above article had already been published elsewhere, or were already under consideration for publication, prior to its submission to Oncology Reports, the Editor has decided that this paper should be retracted from the Journal. The authors independently contacted the Editorial Office requesting that the paper be retracted. The Editor apologizes to the readership for any inconvenience caused. [the original article was published in Oncology Reports 34: 979­986, 2015; DOI: 10.3892/or.2015.4013].

12.
Transl Androl Urol ; 10(9): 3566-3578, 2021 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-34733653

RESUMEN

BACKGROUND: Urine-derived stem cells (USCs) have been widely researched as a novel cell source for stem cell therapy, but their immunomodulatory characteristics remain to be investigated. This study aimed to characterize the immunomodulatory properties of human USCs. METHODS: Human USCs were isolated from fresh voiding urine samples from healthy male donors and expanded. Their cell surface markers were characterized by flow cytometry analysis and the telomerase activities for several USCs clones were determined. The immunosuppressive potential of USCs was evaluated by the performing the mixed lymphocyte reaction (MLR) [co-culture with peripheral blood mononuclear cells (PBMNCs)] and natural killer cells (NK) cytotoxicity assay. USCs cytokines release profile was determined by using human cytokine proteome array. RESULTS: USCs exhibited high cell surface expression of embryonic/mesenchymal stem cells (MSCs) markers CD29, CD44, CD54, CD73, CD90, CD146, and CD166, while lacked expression of hematopoietic stem cell markers CD11, CD14, CD19, CD31, CD34, CD45, B cell marker CD79, and co-stimulatory factors CD80 and CD86, thus, exhibiting the phenotype of MSCs. MLR indicated that USCs significantly inhibited the proliferation of PBMNCs, as compared to that of the human smooth muscle cells (SMCs). In cell cytotoxicity assays, NK cells displayed less cytotoxicity against USCs than against bone marrow mesenchymal stem cells (BMSCs) and SMCs. Furthermore, upon PBMNCs stimulation, USCs secreted higher levels of immunomodulatory cytokines, including IL-6, IL-8, MCP-1, RANTES, GROα, and GM-CSF, compared to those of BMSCs, especially when directly contact mix-culture with PBMNCs. CONCLUSIONS: USCs secreted immunoregulatory cytokines and possessed immunomodulatory properties, comparable to those of BMSCs.

13.
Front Genet ; 12: 600632, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34262589

RESUMEN

As multicellular organisms age, they undergo a reduction in tissue and organ function. Researchers have put forward a theory that stem cell aging is the main factor responsible for decreased tissue and organ function. The adult stem cells guarantee the maintenance and repair of adult tissues and organs. Among adult stem cells, mesenchymal stem cells (MSCs) are emerging as hopeful candidates for cell-based therapy of numerous diseases. In recent years, high-throughput sequencing technologies have evolved to identify circular RNAs (circRNAs) associated with an increasing number of diseases, such as cancer and age-related diseases. It has been reported that circRNAs can compete with microRNAs (miRNAs) to affect the stability or translation of target RNAs and further regulate gene expression at the transcriptional level. However, the role of circRNAs expressed in MSCs in aging mechanisms has not yet been deciphered. The aim of this study was to explore and analyze the expression profiles of age-related circRNAs in MSCs. In this study, bone marrow MSCs were extracted from aged and young rats and analyzed using high-throughput sequencing and bioinformatics. The reliability of high-throughput RNA sequencing was verified by quantitative real-time polymerase chain reaction. The most important circRNA functions and pathways were further selected by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomics (KEGG) analysis. Age-related circRNAs were found in the circrNA-miRNA-mRNA interaction network. The results of high-throughput sequencing showed that 4,229 circRNAs were involved in age-related senescence of MSCs. Compared with the young group, there were 29 differentially expressed circRNAs in the aged group, of which four were upregulated and 25 were downregulated. GO analysis covered three domains: biological process (BP), cellular component (CC), and molecular function (MF). The terms assigned to the BP domain were cellular metabolic processes and cellular macromolecule metabolic processes. The identified CC terms were intracellular and intracellular part, and the identified MF terms were binding and protein binding. The top five KEGG pathways were mitophagy-animal-Rattus norvegicus, prostate cancer-Rattus norvegicus, pathways in cancer-Rattus norvegicus, lysosome-Rattus norvegicus, and autophagy-animal-Rattus norvegicus. Altogether, circRNAs may play a major role in age-related MSC senescence. This study provides new mechanistic insights into MSC senescence, possibly leading to novel therapeutic strategies for age-related diseases.

14.
Stem Cell Res Ther ; 12(1): 271, 2021 05 06.
Artículo en Inglés | MEDLINE | ID: mdl-33957971

RESUMEN

BACKGROUND: Expansion-mediated replicative senescence and age-related natural senescence have adverse effects on mesenchymal stem cell (MSC) regenerative capability and functionality, thus severely impairing the extensive applications of MSC-based therapies. Emerging evidences suggest that microRNA-34a (miR-34a) has been implicated in the process of MSC senescence; however, the molecular mechanisms with regard to how miR-34a influencing MSC senescence remain largely undetermined. METHODS: MiR-34a expression in MSCs was evaluated utilizing RT-qPCR. The functional effects of miR-34a exerting on MSC senescence were investigated via gene manipulation. Relevant gene and protein expression levels were analyzed by RT-qPCR and western blot. Luciferase reporter assays were applied to confirm that Nampt is a direct target of miR-34a. The underlying regulatory mechanism of miR-34a targeting Nampt in MSC senescence was further explored by measuring intracellular NAD+ content, NAD+/NADH ratio and Sirt1 activity. RESULTS: In contrast to Nampt expression, miR-34a expression incremented in senescent MSCs. MiR-34a overexpression in young MSCs resulted in senescence-associated characteristics as displayed by senescence-like morphology, prolonged cell proliferation, declined osteogenic differentiation potency, heightened senescence-associated-ß-galactosidase activity, and upregulated expression levels of the senescence-associated factors. Conversely, miR-34a suppression in replicative senescent and natural senescent MSCs contributed to diminished senescence-related phenotypic features. We identified Nampt as a direct target gene of miR-34a. In addition, miR-34a repletion resulted in prominent reductions in Nampt expression levels, NAD+ content, NAD+/NADH ratio, and Sirt1 activity, whereas anti-miR-34a treatment exerted the opposite effects. Furthermore, miR-34a-mediated MSC senescence was evidently rescued following the co-treatment with Nampt overexpression. CONCLUSION: This study identifies a significant role of miR-34a playing in MSC replicative senescence and natural senescence via targeting Nampt and further mediating by NAD+-Sirt1 pathway, carrying great implications for optimal strategies for MSC therapeutic applications.


Asunto(s)
Células Madre Mesenquimatosas , MicroARNs , Senescencia Celular , MicroARNs/genética , NAD , Osteogénesis , Sirtuina 1/genética
15.
Zhonghua Bing Li Xue Za Zhi ; 39(3): 187-91, 2010 Mar.
Artículo en Zh | MEDLINE | ID: mdl-20450767

RESUMEN

OBJECTIVE: To investigate the biology of HeLa cells upon inhibition of human telomerase catalytic subunit (hTERT) gene by RNA interference in vitro. METHODS: Four shRNAs (A, B, C and D) targeting hTERT gene were designed and prepared by in-vitro transcription. The expression of hTERT gene was evaluated by immunofluorescent staining and telomeric repeat amplification protocol (TRAP) ELISA (TRAP-ELISA), after transient transfection of shRNAs by lipid formulation. Through the initial selection, shRNA (B) was noticed as the most efficient one in down-regulating hTERT gene and therefore was chosen as the ultimate shRNA used in the experimental groups. Those transfected by non-silencing RNAi were chosen as the control groups. Cell spreading and migration were studied by microscopy and cell adhesion to fibronectin (FN) was assayed by cell counting kit-8 (CCK-8). Cell invasion was assessed by Boyden chamber assay. RESULTS: Cell spreading study revealed that the rates of spreading cells in the experimental groups were (5.6 +/- 2.3)% at 30 min, and (26.3 +/- 6.1)% 2 h after the inoculation, respectively, whereas the rates of spreading cells in the control groups were (31.3 +/- 7.9)% and (79.4 +/- 4.8)%, respectively. There were significant differences between the two groups (P < 0.01). However, most of the cells in both groups became spreading after 24 h. Cell adhesion assay demonstrated that the rate of adhesion cells on FN in experimental groups was (67.2 +/- 2.8)%, less than that in control groups (83.7 +/- 5.4)% (P < 0.05). The relative migration distance was (27.1 +/- 6.2)% in the experimental group, lower than that of the control group (58.7 +/- 15.0)%. The invasion assay revealed that the invading cells were 75.7 +/- 14.5 in the experimental group, in contrast to 165.1 +/- 11.0 in the control group after 4 h incubation on matrigel. The difference between these two groups was significant (P < 0.05). CONCLUSION: In vitro shRNA silencing of hTERT gene can down-regulate the telomerase activity, leading to an inhibition of the malignant phenotype of HeLa cells, including decreased ability of cell spreading and adhesion, reduction of cell migration, and declined invasive ability through Matrige assay.


Asunto(s)
Adhesión Celular , Movimiento Celular , Proliferación Celular , Interferencia de ARN , ARN Interferente Pequeño/farmacología , Telomerasa/metabolismo , Regulación hacia Abajo , Regulación Neoplásica de la Expresión Génica , Células HeLa , Humanos , Invasividad Neoplásica , Telomerasa/genética , Transfección
16.
Front Cell Dev Biol ; 8: 599376, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-33330487

RESUMEN

Oxidative stress is one of the main causes of aging. The process of physiological aging is always accompanied by increased levels of endogenous oxidative stress. Exogenous oxidants have contributed to premature cellular senescence. As a deacetylase located in mitochondrial matrix, Sirt3 plays critical roles in mitochondrial energy metabolism, oxidative stress regulation, and cellular senescence. However, it remains unknown whether Sirt3 exerts the analogous role in cellular senescence caused by two different oxidation pathways. In this study, the function of Sirt3 was investigated in age-related natural senescence and H2O2-induced premature senescence of rat bone marrow mesenchymal stem cells (MSCs). Our results showed that Sirt3 expression was significantly decreased in both senescent MSCs, which was concerned with reduced cellular reactive oxygen species (ROS) and aggravated DNA injury. Sirt3 repletion could partly reverse the senescence-associated phenotypic features in natural and premature senescent MSCs. Moreover, Sirt3 replenishment led to the reduction in the levels of cellular ROS by enhancing the expression and activity of superoxide dismutase 2 (SOD2), thus maintaining the balance of intracellular oxidation and antioxidation and ameliorating oxidative stress damage. Altogether, Sirt3 inhibits MSC natural senescence and H2O2-induced premature senescence through alleviating ROS-induced injury and upregulating SOD2 expression and activity. Our research indicates that Sirt3 might contribute to uncovering the novel mechanisms underlying MSC senescence and provide new insights to aging and oxidative stress-related diseases.

17.
J Diabetes Res ; 2020: 4981814, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32083135

RESUMEN

As a chronic metabolic disease, diabetes mellitus (DM) is broadly characterized by elevated levels of blood glucose. Novel epidemiological studies demonstrate that some diabetic patients have an increased risk of developing dementia compared with healthy individuals. Alzheimer's disease (AD) is the most frequent cause of dementia and leads to major progressive deficits in memory and cognitive function. Multiple studies have identified an increased risk for AD in some diabetic populations, but it is still unclear which diabetic patients will develop dementia and which biological characteristics can predict cognitive decline. Although few mechanistic metabolic studies have shown clear pathophysiological links between DM and AD, there are several plausible ways this may occur. Since AD has many characteristics in common with impaired insulin signaling pathways, AD can be regarded as a metabolic disease. We conclude from the published literature that the body's diabetic status under certain circumstances such as metabolic abnormalities can increase the incidence of AD by affecting glucose transport to the brain and reducing glucose metabolism. Furthermore, due to its plentiful lipid content and high energy requirement, the brain's metabolism places great demands on mitochondria. Thus, the brain may be more susceptible to oxidative damage than the rest of the body. Emerging evidence suggests that both oxidative stress and mitochondrial dysfunction are related to amyloid-ß (Aß) pathology. Protein changes in the unfolded protein response or endoplasmic reticulum stress can regulate Aß production and are closely associated with tau protein pathology. Altogether, metabolic disorders including glucose/lipid metabolism, oxidative stress, mitochondrial dysfunction, and protein changes caused by DM are associated with an impaired insulin signal pathway. These metabolic factors could increase the prevalence of AD in diabetic patients via the promotion of Aß pathology.


Asunto(s)
Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Diabetes Mellitus/metabolismo , Glucosa/metabolismo , Insulina/metabolismo , Mitocondrias/metabolismo , Enfermedad de Alzheimer/epidemiología , Glucemia , Diabetes Mellitus/epidemiología , Estrés del Retículo Endoplásmico , Proteínas Facilitadoras del Transporte de la Glucosa/metabolismo , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Humanos , Metabolismo de los Lípidos , Estrés Oxidativo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor de Insulina/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismo , Respuesta de Proteína Desplegada
18.
Acta Biomater ; 107: 50-64, 2020 04 15.
Artículo en Inglés | MEDLINE | ID: mdl-32044457

RESUMEN

Traditional cell therapy technology relies on the maximum expansion of primary stem cells in vitro, through multiple passages and potential differentiation protocols, in order to generate the abundance of cells needed prior to transplantation in vivo. Implantation of in vitro over-expanded and pre-differentiated cells typically results in poor cell survival and reduced regeneration capacity for tissue repair in vivo. We hypothesized that implantation of primary stem cells, after a short time culture in vitro (passage number ≤p3), in combination with controlled release of relevant growth factors would improve in vivo cell viability, engraftment and tissue regeneration. The goal of this study was to determine whether the release of myogenic growth factors from a heparin-hyaluronic acid gel (hp-HA gel) could enhance in vivo cell survival, in-growth and myogenic differentiation of human urine-derived stem cells (USC) with a corresponding enhancement in graft vascularization, innervation and regenerative properties. Human USC were obtained from healthy adult donors (n = 6), expanded and then mixed with a hp-HA gel containing sets of growth factors known to enhance myogenesis (IGF1, HGF, PDGF-BB), neurogenesis (NGF, FGF) and angiogenesis (VEGF), or a cocktail with a combination of growth factors. Primary cultured USC (p3) mixed with the hp-HA gel and the various combinations of growth factors, were subcutaneously injected into athymic mice. In vivo cell survival, engraftment and functional differentiation within the host tissue were assessed. The implanted grafts containing USC and the growth factor cocktail showed the greatest number of surviving cells as well as increased numbers of cells that expressed myogenic and endothelial cell markers as compared to other groups 4 weeks after implantation. Moreover, the graft with USC and the growth factor cocktail showed increased numbers of blood vessels and infiltrating neurons. Thus, growth factors released in a controlled manner from an hp-HA gel containing USC efficiently improved in vivo cell survival and supported vascularization and myogenic differentiation within the grafts. This study provides evidence for the use of primary USC and growth factors in a hydrogel as a novel mode of cell therapy for the promotion of myogenic differentiation for the treatment of injured muscle tissue. STATEMENT OF SIGNIFICANCE: Cell therapies are a promising treatment option for neuromuscular dysfunction disorders. However, major limitations in cell retention and engraftment after implantation remain a hindrance to the use of stem cell therapy for the treatment of muscle injuries or diseased tissues. Implanted long-term in vitro cultured cells tend to demonstrate low rates of survival and tissue engraftment, lessened paracrine effects, and poor homing and differentiation. Human USC are an easily obtainable stem cell source that possess stem cell characteristics such as a robust proliferative potential, paracrine effects on neighboring cells, and multi-potential differentiation. In this study, we demonstrated that a combination of primary human USC with a cocktail of growth factors combined in a hyaluronic gel was optimal for cell survival and engraftment, including myogenic differentiation potential of USC, angiogenesis and host nerve fiber recruitment in vivo. The present study also demonstrated that the use of primary urine derived stem cells at early passages, without in vitro pre-differentiation, implanted in a hyaluronic-heparin hydrogel containing a cocktail of growth factors, provided an alternative safe site-specific delivery method for cell therapy.


Asunto(s)
Células Madre Adultas/efectos de los fármacos , Heparina/química , Ácido Hialurónico/química , Hidrogeles/química , Péptidos y Proteínas de Señalización Intercelular/uso terapéutico , Desarrollo de Músculos/efectos de los fármacos , Adulto , Células Madre Adultas/trasplante , Animales , Diferenciación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Sistemas de Liberación de Medicamentos , Humanos , Masculino , Ratones Desnudos , Neovascularización Fisiológica/efectos de los fármacos , Regeneración Nerviosa/efectos de los fármacos , Trasplante de Células Madre/métodos , Adulto Joven
19.
Aging (Albany NY) ; 11(11): 3505-3522, 2019 06 07.
Artículo en Inglés | MEDLINE | ID: mdl-31175267

RESUMEN

In vitro replicative senescence affects MSC characteristics and functionality, thus severely restricting their application in regenerative medicine and MSC-based therapies. Previously, we found that MSC natural senescence is accompanied by altered intracellular nicotinamide adenine dinucleotide (NAD+) metabolism, in which Nampt plays a key role. However, whether Nampt influences MSC replicative senescence is still unclear. Our study showed that Nampt expression is down-regulated during MSC replicative senescence. Nampt depletion via a specific Nampt inhibitor FK866 or Nampt knockdown in early passage MSCs led to enhanced senescence as indicated by senescence-like morphology, reduced proliferation, and adipogenic and osteogenic differentiation, and increased senescence-associated-ß-galactosidase activity and the expression of the senescence-associated factor p16INK4a. Conversely, Nampt overexpression ameliorated senescence-associated phenotypic features in late passage MSCs. Further, Nampt inhibition resulted in reduced intracellular NAD+ content, NAD+/NADH ratio, and Sirt1 activity, whereas overexpression had the opposite effects. Exogenous intermediates involved in NAD+ biosynthesis not only rescued replicative senescent MSCs but also alleviated FK866-induced MSC senescence. Thus, Nampt suppresses MSC senescence via mediating NAD+-Sirt1 signaling. This study provides novel mechanistic insights into MSC replicative senescence and a promising strategy for the severe shortage of cells for MSC-based therapies.


Asunto(s)
Senescencia Celular/fisiología , Células Madre Mesenquimatosas/metabolismo , NAD/metabolismo , Nicotinamida Fosforribosiltransferasa/metabolismo , Transducción de Señal/fisiología , Sirtuina 1/metabolismo , Acrilamidas/farmacología , Animales , Senescencia Celular/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Masculino , Células Madre Mesenquimatosas/efectos de los fármacos , Nicotinamida Fosforribosiltransferasa/antagonistas & inhibidores , Nicotinamida Fosforribosiltransferasa/genética , Piperidinas/farmacología , Ratas , Ratas Wistar , Transducción de Señal/efectos de los fármacos
20.
Zhonghua Bing Li Xue Za Zhi ; 37(5): 323-7, 2008 May.
Artículo en Zh | MEDLINE | ID: mdl-18956651

RESUMEN

OBJECTIVE: To investigate the telomerase activity and to document biological behaviors of HeLa cells upon treatment with specific PI3K/AKT signaling pathway inhibitor, LY294002. METHODS: CCK-8 assay was used to determine IC50 of LY294002. The expressions of total AKT and phosphorylation AKT (P-AKT) were determined using Western blot. Telomerase activity of cell was measured by TRAP-ELISA assay. Cell growth curve, flow cytometry technique and Hoechst33258 stain were used to evaluate the cell growth, cell cycle and apoptosis respectively. Cell migration was determined by cell wound healing assay. RESULTS: IC50 value of LY294002 of treated HeLa cells was 1.73 mg/L. Western blot showed that LY294002 enabled to decrease P-AKT activity in the presence of same total AKT protein. The cell telomerase activity was decreased to 36.72% in contrast to 98.61% of the control. LY294002 decreased the telomerase activity in HeLa cells, and the growth capacity of the cells was significantly suppressed. The number of cells at G0/G1 phases increased to 66.88% compared with that of the control cells (47.36%). The apoptosis rate also increased from 2.4% to 14.9%. The relative migration distance decreased to 24.6% compared with that of control (62.57%). CONCLUSION: LY294002 inhibition of PI3K/AKT signaling pathway leads to alteration of telomerase activity along with changes of the biological behaviors of the HeLa cells suggesting that regulation of telomerase activity may be closely related to PI3K/AKT signaling pathway.


Asunto(s)
Apoptosis/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Cromonas/farmacología , Morfolinas/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Transducción de Señal/efectos de los fármacos , Western Blotting , Línea Celular , Movimiento Celular/fisiología , Inhibidores Enzimáticos/farmacología , Fase G1/efectos de los fármacos , Células HeLa , Humanos , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt , Transducción de Señal/fisiología , Telomerasa
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