RESUMO
Acute skeletal muscle injury initiates a process of necrosis, debris clearance, and ultimately tissue regeneration via myogenesis. While skeletal muscle stem cells (MuSCs) are responsible for populating the proliferative myogenic progenitor pool to fuel muscle repair, recruited and resident immune cells have a central role in the regulation of muscle regeneration via the execution of phagocytosis and release of soluble factors that act directly on MuSCs to regulate myogenic differentiation. Therefore, the timing of MuSC proliferation and differentiation is closely linked to the populations and behaviors of immune cells present within skeletal muscle. This has important implications for aging and muscle repair, as systemic changes in immune system function contribute to a decline in muscle regenerative capacity. Here, we present adapted protocols for the isolation of mononuclear cells from skeletal muscles for the quantification of immune cell populations using flow cytometry. We also describe a cardiotoxin skeletal muscle injury protocol and detail the expected outcomes including immune cell infiltration to the injured sites and formation of new myocytes. As immune cell function is substantially influenced by aging, we extend these approaches and outcomes to aged mice.
Assuntos
Envelhecimento , Modelos Animais de Doenças , Músculo Esquelético , Regeneração , Animais , Camundongos , Músculo Esquelético/lesões , Músculo Esquelético/metabolismo , Envelhecimento/fisiologia , Desenvolvimento Muscular , Citometria de Fluxo/métodos , Diferenciação Celular , Proliferação de CélulasRESUMO
Currently, mitochondrial dysfunction caused by oxidative stress is a growing concern in degenerative diseases, notably intervertebral disc degeneration (IVDD). Dysregulation of the balance of mitochondrial quality control (MQC) has been considered the key contributor, while it's still challenging to effectively harmonize different MQC components in a simple and biologically safe way. Hydrogen gas (H2) is a promising mitochondrial therapeutic molecule due to its bio-reductivity and diffusibility across cellular membranes, yet its relationship with MQC regulation remains unknown. Herein, we propose a mitochondrial 'Birth-Death' coordinator achieved by an intelligent hydrogen nanogenerator (Fe@HP-OD), which can sustainably release H2 in response to the unique microenvironment in degenerated IVDs. Both in vitro and in vivo results prove alleviation of cellular oxidative stress and restoration of nucleus pulposus cells function, thereby facilitating successful IVD regeneration. Significantly, this study for the first time proposes the mitochondrial 'Birth-Death' coordination mechanism: 1) attenuation of overactivated mitochondrial 'Death' process (UPRmt and unselective mitophagy); and 2) activation of Adenosine 5'-monophosphate-activated protein kinase (AMPK) signaling pathway for mitochondrial 'Birth-Death' balance (mitochondrial biogenesis and controlled mitophagy). These pioneering findings can fill in the gaps in molecular mechanisms for H2 regulation on MQC homeostasis, and pave the way for future strategies towards restoring equilibrium of MQC system against degenerative diseases.
Assuntos
Hidrogênio , Degeneração do Disco Intervertebral , Mitocôndrias , Estresse Oxidativo , Hidrogênio/química , Animais , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Regeneração/efeitos dos fármacos , Disco Intervertebral/efeitos dos fármacos , Humanos , Mitofagia/efeitos dos fármacos , Ratos Sprague-Dawley , Masculino , Núcleo Pulposo/metabolismo , RatosRESUMO
Mesenchymal stem cells (MSCs) are expected to be useful therapeutics in osteoarthritis (OA), the most common joint disorder characterized by cartilage degradation. However, evidence is limited with regard to cartilage repair in clinical trials because of the uncontrolled differentiation and weak cartilage-targeting ability of MSCs after injection. To overcome these drawbacks, here we synthesized CuO@MSN nanoparticles (NPs) to deliver Sox9 plasmid DNA (favoring chondrogenesis) and recombinant protein Bmp7 (inhibiting hypertrophy). After taking up CuO@MSN/Sox9/Bmp7 (CSB NPs), the expressions of chondrogenic markers were enhanced while hypertrophic markers were decreased in response to these CSB-engineered MSCs. Moreover, a cartilage-targeted peptide (designated as peptide W) was conjugated onto the surface of MSCs via a click chemistry reaction, thereby prolonging the residence time of MSCs in both the knee joint cavity of mice and human-derived cartilage. In a surgery-induced OA mouse model, the NP and peptide dual-modified W-CSB-MSCs showed an enhancing therapeutic effect on cartilage repair in knee joints compared with other engineered MSCs after intra-articular injection. Most importantly, W-CSB-MSCs accelerated cartilage regeneration in damaged cartilage explants derived from OA patients. Thus, this new peptide and NPs dual engineering strategy shows potential for clinical applications to boost cartilage repair in OA using MSC therapy.
Assuntos
Diferenciação Celular , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais , Nanopartículas , Osteoartrite , Peptídeos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Animais , Osteoartrite/terapia , Osteoartrite/patologia , Nanopartículas/química , Humanos , Diferenciação Celular/efeitos dos fármacos , Peptídeos/química , Transplante de Células-Tronco Mesenquimais/métodos , Condrogênese/efeitos dos fármacos , Camundongos , Fatores de Transcrição SOX9/metabolismo , Fatores de Transcrição SOX9/genética , Cartilagem Articular/patologia , Cartilagem Articular/efeitos dos fármacos , Proteína Morfogenética Óssea 7/química , Proteína Morfogenética Óssea 7/farmacologia , Engenharia Tecidual/métodos , Regeneração/efeitos dos fármacosRESUMO
The unsuitable deformation stimulus, harsh urine environment, and lack of a regenerative microenvironment (RME) prevent scaffold-based urethral repair and ultimately lead to irreversible urethral scarring. The researchers clarify the optimal elastic modulus of the urethral scaffolds for urethral repair and design a multilayered PVA hydrogel scaffold for urethral scar-free healing. The inner layer of the scaffold has self-healing properties, which ensures that the wound effectively resists harsh urine erosion, even when subjected to sutures. In addition, the scaffold's outer layer has an extracellular matrix-like structure that synergizes with adipose-derived stem cells to create a favorable RME. In vivo experiments confirm successful urethral scar-free healing using the PVA multilayered hydrogel scaffold. Further mechanistic study shows that the PVA multilayer hydrogel effectively resists the urine-induced inflammatory response and accelerates the transition of urethral wound healing to the proliferative phase by regulating macrophage polarization, thus providing favorable conditions for urethral scar-free healing. This study provides mechanical criteria for the fabrication of urethral tissue-engineered scaffolds, as well as important insights into their design.
Assuntos
Módulo de Elasticidade , Hidrogéis , Alicerces Teciduais , Uretra , Cicatrização , Alicerces Teciduais/química , Animais , Hidrogéis/química , Engenharia Tecidual/métodos , Camundongos , Regeneração , Cicatriz/patologia , Masculino , Microambiente Celular , Ratos Sprague-Dawley , Células-Tronco/citologiaRESUMO
Several protocols have been established for the generation of lens organoids from embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and other cells with regenerative potential in humans or various animal models. It is important to examine how well the regenerated lens organoids reflect lens biology, in terms of its development, homeostasis, and aging. Toward this goal, the iSyTE database (integrated Systems Tool for Eye gene discovery; https://research.bioinformatics.udel.edu/iSyTE/ ), a bioinformatics resource tool that contains meta-analyzed gene expression data in wild-type lens across different embryonic, postnatal, and adult stages, can serve as a resource for comparative analysis. This article outlines the approaches toward effective use of iSyTE to gain insights into normal gene expression in the mouse lens, enriched expression in the lens, and differential gene expression in select mouse gene-perturbation cataract/lens defects models, which in turn can be used to evaluate expression of key lens-relevant genes in lens organoids by transcriptomics (e.g., RNA-sequencing (RNA-seq), microarrays, etc.) or other downstream methods (e.g., RT-qPCR, etc.).
Assuntos
Cristalino , Organoides , Regeneração , Cristalino/citologia , Cristalino/metabolismo , Organoides/metabolismo , Organoides/citologia , Animais , Camundongos , Regeneração/genética , Perfilação da Expressão Gênica/métodos , Biologia Computacional/métodos , Simulação por Computador , Humanos , Catarata/genética , Catarata/patologia , Catarata/metabolismo , Transcriptoma , Bases de Dados GenéticasRESUMO
Retinal degenerative diseases including age-related macular degeneration and glaucoma are estimated to currently affect more than 14 million people in the United States, with an increased prevalence of retinal degenerations in aged individuals. An expanding aged population who are living longer forecasts an increased prevalence and economic burden of visual impairments. Improvements to visual health and treatment paradigms for progressive retinal degenerations slow vision loss. However, current treatments fail to remedy the root cause of visual impairments caused by retinal degenerations-loss of retinal neurons. Stimulation of retinal regeneration from endogenous cellular sources presents an exciting treatment avenue for replacement of lost retinal cells. In multiple species including zebrafish and Xenopus, Müller glial cells maintain a highly efficient regenerative ability to reconstitute lost cells throughout the organism's lifespan, highlighting potential therapeutic avenues for stimulation of retinal regeneration in humans. Here, we describe how the application of single-cell RNA-sequencing (scRNA-seq) has enhanced our understanding of Müller glial cell-derived retinal regeneration, including the characterization of gene regulatory networks that facilitate/inhibit regenerative responses. Additionally, we provide a validated experimental framework for cellular preparation of mouse retinal cells as input into scRNA-seq experiments, including insights into experimental design and analyses of resulting data.
Assuntos
Células Ependimogliais , Retina , Análise de Célula Única , Animais , Camundongos , Análise de Célula Única/métodos , Retina/metabolismo , Células Ependimogliais/metabolismo , Regeneração/genética , Análise de Sequência de RNA/métodos , Degeneração Retiniana/genética , Degeneração Retiniana/terapia , RNA-Seq/métodos , Modelos Animais de DoençasRESUMO
Glaucoma is one of the leading causes of irreversible blindness. Stem cell therapy has shown promise in the treatment of primary open-angle glaucoma in animal models. Stem cell-free therapy using stem cell-derived trophic factors might be in demand in patients with high-risk conditions or religious restrictions. In this chapter, we describe methods for trabecular meshwork stem cell (TMSC) cultivation, secretome harvesting, and protein isolation, as well as assays to ensure the health of TMSC post-secretome harvesting and for secretome periocular injection into mice for therapeutic purposes.
Assuntos
Células-Tronco , Malha Trabecular , Malha Trabecular/metabolismo , Malha Trabecular/citologia , Animais , Camundongos , Humanos , Células-Tronco/citologia , Células-Tronco/metabolismo , Regeneração , Glaucoma/terapia , Transplante de Células-Tronco/métodos , Secretoma , Modelos Animais de Doenças , Glaucoma de Ângulo Aberto/terapia , Células Cultivadas , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Técnicas de Cultura de Células/métodosRESUMO
Mammals do not possess the ability to spontaneously repair or regenerate damaged retinal tissue. In contrast to teleost fish which are capable of retina regeneration through the action of Müller glia, mammals undergo a process of reactive gliosis and scarring that inhibits replacement of lost neurons. Thus, it is important to discover novel methods for stimulating mammalian Müller glia to dedifferentiate and produce progenitor cells that can replace lost retinal neurons. Inducing an endogenous regenerative pathway mediated by Müller glia would provide an attractive alternative to stem cell injections or gene therapy approaches. Extracellular vesicles (EVs) are now recognized to serve as a novel form of cell-cell communication through the transfer of cargo from donor to recipient cells or by the activation of signaling cascades in recipient cells. EVs have been shown to promote proliferation and regeneration raising the possibility that delivery of EVs could be a viable treatment for visual disorders. Here, we provide protocols to isolate EVs for use in retina regeneration experiments.
Assuntos
Vesículas Extracelulares , Regeneração , Retina , Animais , Vesículas Extracelulares/metabolismo , Retina/metabolismo , Retina/citologia , Retina/fisiologia , Células Ependimogliais/metabolismo , Células Ependimogliais/citologia , Camundongos , Comunicação Celular , Proliferação de Células , Regeneração Nervosa/fisiologiaRESUMO
Various strategies for replacing retinal neurons lost in degenerative diseases are under investigation, including stimulating the endogenous regenerative capacity of Müller Glia (MG) as injury-inducible retinal stem cells. Inherently regenerative species, such as zebrafish, have provided key insights into mechanisms regulating MG dedifferentiation to a stem-like state and the proliferation of MG and MG-derived progenitor cells (MGPCs). Interestingly, promoting MG/MGPC proliferation is not sufficient for regeneration, yet mechanistic studies are often focused on this measure. To fully account for the regenerative process, and facilitate screens for factors regulating cell regeneration, an assay for quantifying cell replacement is required. Accordingly, we adapted an automated reporter-assisted phenotypic screening platform to quantify the pace of cellular regeneration kinetics following selective cell ablation in larval zebrafish. Here, we detail a method for using this approach to identify chemicals and genes that control the rate of retinal cell regeneration following selective retinal cell ablation.
Assuntos
Peixe-Zebra , Animais , Retina/citologia , Retina/metabolismo , Fenótipo , Proliferação de Células , Regeneração , Células Ependimogliais/citologia , Células Ependimogliais/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Cinética , Regeneração Nervosa/fisiologiaRESUMO
Controlled release or controlled drug delivery comprises the set of techniques and approaches to improve bioavailability through improved safety and/or efficacy using a carrier material for the molecule of interest. The predictability and tunability of these carriers make them ideal for protection, localization, and sustained presentation of a wide range of therapeutics, including growth factors implicated in cell survival and regeneration. Here we provide a method for encapsulating epidermal growth factor in a degradable polymer matrix for delivery to the cornea. Additional notes are included to demonstrate the wide-ranging capabilities of such methods for other materials, therapeutic agents, and sites of action within the eye.
Assuntos
Sobrevivência Celular , Preparações de Ação Retardada , Sobrevivência Celular/efeitos dos fármacos , Humanos , Regeneração , Fator de Crescimento Epidérmico/metabolismo , Animais , Córnea/metabolismo , Córnea/citologia , Sistemas de Liberação de Medicamentos/métodos , Polímeros/química , Portadores de Fármacos/químicaRESUMO
Unlike mammals, some nonmammalian species recruit Müller glia for retinal regeneration after injury. Identifying the underlying mechanisms may help to foresee regenerative medicine strategies. Using a Xenopus model of retinitis pigmentosa, we found that Müller cells actively proliferate upon photoreceptor degeneration in old tadpoles but not in younger ones. Differences in the inflammatory microenvironment emerged as an explanation for such stage dependency. Functional analyses revealed that enhancing neuroinflammation is sufficient to trigger Müller cell proliferation, not only in young tadpoles but also in mice. In addition, we showed that microglia are absolutely required for the response of mouse Müller cells to mitogenic factors while negatively affecting their neurogenic potential. However, both cell cycle reentry and neurogenic gene expression are allowed when applying sequential pro- and anti-inflammatory treatments. This reveals that inflammation benefits Müller glia proliferation in both regenerative and nonregenerative vertebrates and highlights the importance of sequential inflammatory modulation to create a regenerative permissive microenvironment.
Assuntos
Proliferação de Células , Células Ependimogliais , Regeneração , Retina , Animais , Células Ependimogliais/metabolismo , Células Ependimogliais/patologia , Camundongos , Retina/patologia , Retina/metabolismo , Doenças Neuroinflamatórias/metabolismo , Doenças Neuroinflamatórias/patologia , Doenças Neuroinflamatórias/etiologia , Microglia/metabolismo , Microglia/patologia , Modelos Animais de Doenças , Retinose Pigmentar/patologia , Retinose Pigmentar/metabolismo , Inflamação/patologia , Inflamação/metabolismo , LarvaRESUMO
In lung disease, persistence of KRT8-expressing aberrant basaloid cells in the alveolar epithelium is associated with impaired tissue regeneration and pathological tissue remodeling. We analyzed single cell RNA sequencing datasets of human interstitial lung disease and found the profibrotic Interleukin-11 (IL11) cytokine to be highly and specifically expressed in aberrant KRT8+ basaloid cells. IL11 is similarly expressed by KRT8+ alveolar epithelial cells lining fibrotic lesions in a mouse model of interstitial lung disease. Stimulation of alveolar epithelial cells with IL11 causes epithelial-to-mesenchymal transition and promotes a KRT8-high state, which stalls the beneficial differentiation of alveolar type 2 (AT2)-to-AT1 cells. Inhibition of IL11-signaling in AT2 cells in vivo prevents the accumulation of KRT8+ cells, enhances AT1 cell differentiation and blocks fibrogenesis, which is replicated by anti-IL11 therapy. These data show that IL11 inhibits reparative AT2-to-AT1 differentiation in the damaged lung to limit endogenous alveolar regeneration, resulting in fibrotic lung disease.
Assuntos
Células Epiteliais Alveolares , Diferenciação Celular , Interleucina-11 , Regeneração , Interleucina-11/metabolismo , Interleucina-11/genética , Animais , Regeneração/genética , Humanos , Camundongos , Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/patologia , Alvéolos Pulmonares/patologia , Alvéolos Pulmonares/metabolismo , Transição Epitelial-Mesenquimal/genética , Modelos Animais de Doenças , Doenças Pulmonares Intersticiais/patologia , Doenças Pulmonares Intersticiais/genética , Doenças Pulmonares Intersticiais/metabolismo , Camundongos Endogâmicos C57BL , Masculino , Transdução de Sinais , FemininoRESUMO
BACKGROUND: Embryos are regeneration and wound healing masters. They rapidly close wounds and scarlessly remodel and regenerate injured tissue. Regeneration has been extensively studied in many animal models using new tools such as single-cell analysis. However, until now, they have been based primarily on experiments assessing from 1 day post injury. RESULTS: In this paper, we reveal that critical steps initiating regeneration occur within hours after injury. We discovered the regeneration initiating cells (RICs) using single-cell and spatial transcriptomics of the regenerating Xenopus laevis tail. RICs are formed transiently from the basal epidermal cells, and their expression signature suggests they are important for modifying the surrounding extracellular matrix thus regulating development. The absence or deregulation of RICs leads to excessive extracellular matrix deposition and defective regeneration. CONCLUSION: RICs represent a newly discovered transient cell state involved in the initiation of the regeneration process.
Assuntos
Regeneração , Cauda , Xenopus laevis , Animais , Transcriptoma , Análise de Célula Única , Matriz Extracelular/metabolismo , CicatrizaçãoRESUMO
Background: The musculoskeletal system, due to inherent structure and function, lends itself to contributing toward joint pain, whether from inflammatory disorders such as rheumatoid arthritis, degenerative diseases such as osteoarthritis, or trauma causing soft tissue injury. Administration of peptides for treatment of joint pain or inflammation is an emerging line of therapy that seeks to offer therapeutic benefits while remaining safe and relatively non-invasive. Purpose: The purpose of this study is to review the current literature on existing oral peptide agents, intra-articular peptide agents, and new developments in human trials to assess route of administration (RoA) for drug delivery in terms of soft tissue regeneration. Study Design: Narrative Review. Methods: A comprehensive literature search was conducted using the PubMed database. The search included medical subject headings (MeSH) terms related to peptide therapy, soft tissue regeneration, and RoA. Inclusion criteria comprised articles focusing on the mechanisms of action of peptides, clinical or biochemical outcomes, and review articles. Exclusion criteria included insufficient literature or studies not meeting the set evidence level. Conclusion: The review identified various peptides demonstrating efficacy in soft tissue repair. Oral and intra-articular peptides showed distinct advantages in soft tissue regeneration, with intra-articular routes providing localized effects and oral routes offering systemic benefits. However, both routes have limitations in bioavailability and absorption. Still in their infancy, further inquiries/research into the properties and efficacy of emerging peptides will be necessary before widespread use. As a viable alternative prior to surgical intervention, peptide treatments present as promising candidates for positive outcomes in soft tissue regeneration.
Assuntos
Peptídeos , Regeneração , Humanos , Regeneração/efeitos dos fármacos , Peptídeos/uso terapêutico , Peptídeos/farmacologiaRESUMO
BACKGROUND: The formation of stem cell clones enables close contact of stem cells inside. The gap junctions in such clone spheres establish a microenvironment that allows frequent intercellular communication to maintain self-renewal and functions of stem cells. Nevertheless, the essential gap junction protein for molecular signaling in clones is poorly known. METHODS: Primary human airway basal cells (hBCs) were isolated from brushing samples through bronchoscopy and then cultured. A tightly focused femtosecond laser was used to excite the local Ca2+ in an individual cell to initiate an internal Ca2+ wave in a clone to screen gap junction proteins. Immunoflourescence staining and clonogenicity assay were used to evaluate self-renewal and functions. RNA and protein levels were assessed by PCR and Western blot. Air-liquid interface assay was conducted to evaluate the differentiation potential. A Naphthalene injury mouse model was used to assess the regeneration potential. RESULTS: Herein, we identify Connexin 25 (Cx25) dominates intercellular Ca2+ communications in clones of hBCs in vitro to maintain the self-renewal and pluripotency of them. The self-renewal and in vitro differentiation functions and in vivo regeneration potential of hBCs in an airway damage model are both regulated by Cx25. The abnormal expression of Cx25 is validated in several diseases including IPF, Covid-19 and bronchiectasis. CONCLUSION: Cx25 is essential for hBC clones in maintaining self-renewal and functions of hBCs via gap junctions.
Assuntos
Conexinas , Regeneração , Humanos , Animais , Camundongos , Conexinas/metabolismo , Conexinas/genética , Diferenciação Celular , COVID-19/metabolismo , COVID-19/virologia , COVID-19/patologia , Junções Comunicantes/metabolismo , Autorrenovação Celular , Cálcio/metabolismo , Células Cultivadas , SARS-CoV-2/metabolismo , Masculino , Células-Tronco/metabolismo , Células-Tronco/citologiaRESUMO
The rapid advancement of cell therapies underscores the importance of understanding fundamental cellular attributes. Among these, cell fitness-how transplanted cells adapt to new microenvironments and maintain functional stability in vivo-is crucial. This study identifies a chemical compound, FPH2, that enhances the fitness of human chondrocytes and the repair of articular cartilage, which is typically nonregenerative. Through drug screening, FPH2 was shown to broadly improve cell performance, especially in maintaining chondrocyte phenotype and enhancing migration. Single-cell transcriptomics indicated that FPH2 induced a super-fit cell state. The mechanism primarily involves the inhibition of carnitine palmitoyl transferase I and the optimization of metabolic homeostasis. In animal models, FPH2-treated human chondrocytes substantially improved cartilage regeneration, demonstrating well-integrated tissue interfaces in rats. In addition, an acellular FPH2-loaded hydrogel proved effective in preventing the onset of osteoarthritis. This research provides a viable and safe method to enhance chondrocyte fitness, offering insights into the self-regulatory mechanisms of cell fitness.
Assuntos
Cartilagem Articular , Condrócitos , Regeneração , Condrócitos/metabolismo , Condrócitos/citologia , Condrócitos/efeitos dos fármacos , Animais , Humanos , Cartilagem Articular/metabolismo , Ratos , Osteoartrite/metabolismo , Osteoartrite/terapia , Hidrogéis/química , Movimento Celular/efeitos dos fármacosRESUMO
BACKGROUND: Microspore embryogenesis is a process that produces doubled haploids in tissue culture environments and is widely used in cereal plants. The efficient production of green regenerants requires stresses that could be sensed at the level of glycolysis, followed by the Krebs cycle and electron transfer chain. The latter can be affected by Cu(II) ion concentration in the induction media acting as cofactors of biochemical reactions, indirectly influencing the production of glutathione (GSH) and S-adenosyl-L-methionine (SAM) and thereby affecting epigenetic mechanisms involving DNA methylation (demethylation-DM, de novo methylation-DNM). The conclusions mentioned were acquired from research on triticale regenerants, but there is no similar research on barley. In this way, the study looks at how DNM, DM, Cu(II), SAM, GSH, and ß-glucan affect the ability of green plant regeneration efficiency (GPRE). RESULTS: The experiment involved spring barley regenerants obtained through anther culture. Nine variants (trials) of induction media were created by adding copper (CuSO4: 0.1; 5; 10 µM) and silver salts (AgNO3: 0; 10; 60 µM), with varying incubation times for the anthers (21, 28, and 35 days). Changes in DNA methylation were estimated using the DArTseqMet molecular marker method, which also detects cytosine methylation. Phenotype variability in ß-glucans, SAM and GSH induced by the nutrient treatments was assessed using tentative assignments based on the Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) spectroscopy. The effectiveness of green plant regeneration ranged from 0.1 to 2.91 plants per 100 plated anthers. The level of demethylation ranged from 7.61 to 32.29, while de novo methylation reached values ranging from 6.83 to 32.27. The paper demonstrates that the samples from specific in vitro conditions (trials) formed tight groups linked to the factors contributing to the two main components responsible for 55.05% of the variance (to the first component DNM, DM, to the second component GSH, ß-glucans, Cu(II), GPRE). CONCLUSIONS: We can conclude that in vitro tissue culture conditions affect biochemical levels, DNA methylation changes, and GPRE. Increasing Cu(II) concentration in the IM impacts the metabolism and DNA methylation, elevating GPRE. Thus, changing Cu(II) concentration in the IM is fair to expect to boost GPRE.
Assuntos
Metilação de DNA , Glutationa , Hordeum , S-Adenosilmetionina , Técnicas de Cultura de Tecidos , beta-Glucanas , Hordeum/genética , Hordeum/metabolismo , Hordeum/crescimento & desenvolvimento , Hordeum/efeitos dos fármacos , Metilação de DNA/efeitos dos fármacos , Glutationa/metabolismo , Técnicas de Cultura de Tecidos/métodos , beta-Glucanas/metabolismo , S-Adenosilmetionina/metabolismo , Flores/genética , Flores/crescimento & desenvolvimento , Regeneração/efeitos dos fármacosRESUMO
Kidney injury disrupts the intricate renal architecture and triggers limited regeneration, together with injury-invoked inflammation and fibrosis. Deciphering the molecular pathways and cellular interactions driving these processes is challenging due to the complex tissue structure. Here, we apply single cell spatial transcriptomics to examine ischemia-reperfusion injury in the mouse kidney. Spatial transcriptomics reveals injury-specific and spatially-dependent gene expression patterns in distinct cellular microenvironments within the kidney and predicts Clcf1-Crfl1 in a molecular interplay between persistently injured proximal tubule cells and their neighboring fibroblasts. Immune cell types play a critical role in organ repair. Spatial analysis identifies cellular microenvironments resembling early tertiary lymphoid structures and associated molecular pathways. Collectively, this study supports a focus on molecular interactions in cellular microenvironments to enhance understanding of injury, repair and disease.
Assuntos
Comunicação Celular , Microambiente Celular , Rim , Regeneração , Traumatismo por Reperfusão , Transcriptoma , Animais , Camundongos , Regeneração/genética , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/patologia , Rim/metabolismo , Rim/patologia , Camundongos Endogâmicos C57BL , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/patologia , Masculino , Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Análise de Célula Única , FibroseRESUMO
METHODS: Single-cell transcriptomics and high-throughput transcriptomics were used to screen factors significantly correlated with intervertebral disc degeneration (IDD). Expression changes of CFIm25 were determined via RT-qPCR and Western blot. NP cells were isolated from mouse intervertebral discs and induced to degrade with TNF-α and IL-1ß. CFIm25 was knocked out using CRISPR-Cas9, and CFIm25 knockout and overexpressing nucleus pulposus (NP) cell lines were generated through lentiviral transfection. Proteoglycan expression, protein expression, inflammatory factor expression, cell viability, proliferation, migration, gene expression, and protein expression were analyzed using various assays (alcian blue staining, immunofluorescence, ELISA, CCK-8, EDU labeling, transwell migration, scratch assay, RT-qPCR, Western blot). The GelMA-HAMA hydrogel loaded with APET×2 polypeptide and sgRNA was designed, and its effects on NP regeneration were assessed through in vitro and mouse model experiments. The progression of IDD in mice was evaluated using X-ray, H&E staining, and Safranin O-Fast Green staining. Immunohistochemistry was performed to determine protein expression in NP tissue. Proteomic analysis combined with in vitro and in vivo experiments was conducted to elucidate the mechanisms of hydrogel action. RESULTS: CFIm25 was upregulated in IDD NP tissue and significantly correlated with disease progression. Inhibition of CFIm25 improved NP cell degeneration, enhanced cell proliferation, and migration. The hydrogel effectively knocked down CFIm25 expression, improved NP cell degeneration, promoted cell proliferation and migration, and mitigated IDD progression in a mouse model. The hydrogel inhibited inflammatory factor expression (IL-6, iNOS, IL-1ß, TNF-α) by targeting the p38/NF-κB signaling pathway, increased collagen COLII and proteoglycan Aggrecan expression, and suppressed NP degeneration-related factors (COX-2, MMP-3). CONCLUSION: The study highlighted the crucial role of CFIm25 in IDD and introduced a promising therapeutic strategy using a porous spherical GelMA-HAMA hydrogel loaded with APET×2 polypeptide and sgRNA. This innovative approach offers new possibilities for treating degenerated intervertebral discs.