RESUMO
Our research aimed to provide complete histological, histochemical and ultrastructural features of the lacrimal gland of the one-humped camel (Camelus dromedarius) as well as novel insights into its adaptability to the Egyptian desert. Our study was applied to 20 fresh lacrimal glands collected from 10 camels instantly after their slaughtering. The results revealed that the gland was a compound tubulo-acinar gland, and its acini were enclosed by a thick connective tissue capsule that was very rich in elastic and collagen fibres. The gland acini had irregular lumens and were composed of conical to pyramidal cells. The nuclei of secretory cells were found in the basal part, and the cytoplasm was eosinophilic and granular. The glandular tissue consisted of serous and mucous acini and seromucous secretory cells. Histochemically, there was a significant amount of neutral mucopolysaccharides in the acini in which mucous cells had a significant periodic acid-Schiff (PAS)-positive reaction, whereas seromucous cells had a mild PAS-positive reaction. Ultrastructurally, the lacrimal cells had numerous secretory vesicles with contents of moderately to highly electron-dense cytoplasm. The nuclear envelope consisted of two prominent membranes surrounding the peri-nuclear cisterna. The acinar cells had numerous electron-lucent and moderately electron-dense secretory granules, mainly situated on the apical surface, and secreted their contents into the lumen. The luminal surface of the mucous secretory cells represents the remains of secretory granules discharged by the merocrine mechanism. In conclusion, the mucous secretion is believed to aid in the washing and moistening of the eyeball, particularly in dry, hot and dusty environments.
Assuntos
Camelus , Aparelho Lacrimal , Animais , Camelus/anatomia & histologia , Aparelho Lacrimal/anatomia & histologia , Aparelho Lacrimal/ultraestrutura , Aparelho Lacrimal/citologia , Masculino , Vesículas Secretórias/ultraestrutura , Células Acinares/ultraestrutura , Células Acinares/citologia , Feminino , Microscopia Eletrônica de Transmissão/veterinária , Reação do Ácido Periódico de Schiff/veterináriaRESUMO
Aging is associated with a massive infiltration of T lymphocytes in the lacrimal gland. Here, we aimed to characterize the immune phenotype of aged CD4+ T cells in this tissue as compared with lymphoid organs. To perform this, we sorted regulatory T cells (Tregs, CD4+CD25+GITR+) and non-Tregs (CD4+CD25negGITRneg) in lymphoid organs from female C57BL/6J mice and subjected these cells to an immunology NanoString® panel. These results were confirmed by flow cytometry, live imaging, and tissue immunostaining in the lacrimal gland. Importantly, effector T helper 1 (Th1) genes were highly upregulated on aged Tregs, including the master regulator Tbx21. Among the non-Tregs, we also found a significant increase in the levels of EOMESmed/high, TbetnegIFN-γ+, and CD62L+CD44negCD4+ T cells with aging, which are associated with cell exhaustion, immunopathology, and the generation of tertiary lymphoid tissue. At the functional level, aged Tregs from lymphoid organs are less able to decrease proliferation and IFN-γ production of T responders at any age. More importantly, human lacrimal glands (age range 55-81 years) also showed the presence of CD4+Foxp3+ cells. Further studies are needed to propose potential molecular targets to avoid immune-mediated lacrimal gland dysfunction with aging.
Assuntos
Aparelho Lacrimal , Tecido Linfoide , Linfócitos T Reguladores , Idoso , Idoso de 80 Anos ou mais , Animais , Feminino , Humanos , Camundongos , Interferon gama , Aparelho Lacrimal/citologia , Camundongos Endogâmicos C57BL , Fenótipo , Pessoa de Meia-Idade , Tecido Linfoide/citologiaRESUMO
In the present study, the expression of secretory components and vesicular transport proteins in the canine lacrimal gland was examined and morphometric analysis was performed. The secretory epithelium consists of two types of secretory cells with different morphological features. The secretory cells constituting acinar units (type A cells) exhibited higher levels of glycoconjugates, including ß-GlcNAc, than the other cell type constituting tubular units (type T cells). Immunoblot analysis revealed that antimicrobial proteins, such as lysozyme, lactoferrin and lactoperoxidase, Rab proteins (Rab3d, Rab27a and Rab27b) and soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) proteins (VAMP2, VAMP4, VAMP8, syntaxin-1, syntaxin-4 and syntaxin-6), were expressed at various levels. We immunohistochemically demonstrated that the expression patterns of lysozyme, lactoferrin, Raba27a, Rab27b, VAMP4, VAMP8 and syntaxin-6 differed depending on the secretory cell type. Additionally, in type T cells, VAMP4 was confined to a subpopulation of secretory granules, while VAMP8 was detected in almost all of them. The present study displayed the morphological and histochemical characteristics of the secretory epithelium in the canine lacrimal gland. These findings will help elucidate the species-specific properties of this gland.
Assuntos
Aparelho Lacrimal/metabolismo , Vesículas Secretórias/metabolismo , Animais , Cães , Epitélio/metabolismo , Aparelho Lacrimal/citologia , Masculino , Proteínas SNARE/metabolismoRESUMO
Aquaporins (AQPs) are proteins that selectively transport water across the cell membrane. Although AQPs play important roles in secretion in the lacrimal gland, the expression and localization of AQPs have not been clarified yet. In the current study, we investigated the expression pattern of AQP family members in the murine lacrimal gland during development. Lacrimal gland tissues were harvested from E13.5 and E17.5 murine embryos and from mice 8 weeks of age (adults). Corneal and conjunctival tissues from the latter served as controls. Total RNA was isolated and analyzed for the expression of AQP family members using qPCR. The localization of AQPs in the adult lacrimal gland in adult murine lacrimal glands was also analyzed. Expression of Aqp8 and Aqp9 mRNAs was detected in the adult lacrimal gland but not in the cornea, conjunctiva, or fetal lacrimal gland. AQP8 and AQP9 and α-SMA partially colocalized around the basal regions of the acinar unit. The levels of Aqp3 mRNAs and protein were much lower in the adult lacrimal gland but were readily detected in the adult cornea and conjunctiva. Our study suggests that AQP8 and AQP9 may serve as markers for adult murine lacrimal gland, ductal, and myoepithelial cells.
Assuntos
Aquaporinas/metabolismo , Aparelho Lacrimal/citologia , Fatores Etários , Animais , Aquaporinas/análise , Aquaporinas/fisiologia , Membrana Celular/metabolismo , Túnica Conjuntiva/metabolismo , Córnea/metabolismo , Células Epiteliais/metabolismo , Feminino , Expressão Gênica/genética , Aparelho Lacrimal/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/metabolismo , Transcriptoma/genéticaRESUMO
Purpose: The functional characteristics of receptors that regulate lacrimal gland myoepithelial cells are still somewhat unclear. To date, mainly muscarinic receptors have been of interest; however, further knowledge is needed regarding their expression and functional roles. For this purpose, primary cultures of rat lacrimal gland myoepithelial cells were established and examined functionally. Methods: Rat lacrimal glands were excised, minced, and further digested, yielding mixtures of cells that were seeded in culturing flasks. After 4-6 weeks, primary monocultures of myoepithelial cells were established, verified by immunocytochemistry. The cells were stained for all muscarinic receptor subtypes (M1-M5) and examined functionally regarding intracellular [Ca2+] responses upon activation of muscarinic receptors. For methodological verification, purinergic functional responses were also studied. Results: Expression of muscarinic receptor subtypes M2-M5 was detected, whereas expression of muscarinic M1 receptors could not be shown. Activation of muscarinic receptors by the non-selective muscarinic agonist methacholine (3 × 10-11-10-3 M) did not cause a significant increase in intracellular [Ca2+]. However, activation of purinergic receptors by the non-selective purinergic agonist ATP (10-8-10-3 M) caused a concentration-dependent increase in intracellular [Ca2+] that could be blocked by the P2 antagonists PPADS and suramin. Conclusions: Primary cultures of rat lacrimal gland myoepithelial cells were established that displayed a heterogeneous expression of muscarinic receptors. Purinergic functional responses demonstrated a viable cell population. Upon treatment with methacholine, no significant increase in intracellular [Ca2+] could be detected, indicating that cholinergic activation of myoepithelial cells occurs via other intracellular messengers or is dependent on interaction with other cell types.
Assuntos
Células Epiteliais/metabolismo , Aparelho Lacrimal/metabolismo , Receptores Muscarínicos/metabolismo , Receptores Purinérgicos/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Cálcio/metabolismo , Células Cultivadas , Relação Dose-Resposta a Droga , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Imuno-Histoquímica , Aparelho Lacrimal/citologia , Aparelho Lacrimal/efeitos dos fármacos , Masculino , Cloreto de Metacolina/farmacologia , Agonistas Muscarínicos/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
Mesenchymal stem cells (MSCs) have been widely used in therapeutic applications for many decades. However, more and more evidence suggests that factors such as the site of origin and pre-implantation treatment have a crucial impact on the result. This study investigates the role of freshly isolated MSCs in the lacrimal gland after allogeneic transplantation. For this purpose, MSCs from transgenic GFP mice were isolated and transplanted into allogeneic and syngeneic recipients. While the syngeneic MSCs maintained a spherical shape, allogeneic MSCs engrafted into the tissue as spindle-shaped cells in the interstitial stroma. Furthermore, the MSCs produced collagen type I in more than 85% to 95% of the detected GFP+ MSCs in the recipients of both models, supposedly contributing to pathogenic fibrosis in allogeneic recipients compared to syngeneic models. These findings indicate that allogeneic MSCs act completely differently from syngeneic MSCs, highlighting the importance of understanding the exact mechanisms behind MSCs.
Assuntos
Transplante de Medula Óssea , Colágeno Tipo I/biossíntese , Células-Tronco Mesenquimais/metabolismo , Animais , Aparelho Lacrimal/citologia , Células-Tronco Mesenquimais/citologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fenótipo , Transplante Homólogo , Transplante IsogênicoRESUMO
Altered lacrimal gland (LG) secretion is a feature of autoimmune dacryoadenitis in Sjögren's syndrome (SS). Cathepsin S (CTSS) is increased in tears of SS patients, which may contribute to disease. Rab3D and Rab27a/b isoforms are effectors of exocytosis in LG, but Rab27a is poorly studied. To investigate whether Rab27a mediates CTSS secretion, we utilized quantitative confocal fluorescence microscopy of LG from SS-model male NOD and control male BALB/c mice, showing that Rab27a-enriched vesicles containing CTSS were increased in NOD mouse LG. Live-cell imaging of cultured lacrimal gland acinar cells (LGAC) transduced with adenovirus encoding wild-type (WT) mCFP-Rab27a revealed carbachol-stimulated fusion and depletion of mCFP-Rab27a-enriched vesicles. LGAC transduced with dominant-negative (DN) mCFP-Rab27a exhibited significantly reduced carbachol-stimulated CTSS secretion by 0.5-fold and ß-hexosaminidase by 0.3-fold, relative to stimulated LGAC transduced with WT mCFP-Rab27a. Colocalization of Rab27a and endolysosomal markers (Rab7, Lamp2) with the apical membrane was increased in both stimulated BALB/c and NOD mouse LG, but the extent of colocalization was much greater in NOD mouse LG. Following stimulation, Rab27a colocalization with endolysosomal membranes was decreased. In conclusion, Rab27a participates in CTSS secretion in LGAC though the major regulated pathway, and through a novel endolysosomal pathway that is increased in SS.
Assuntos
Catepsinas/metabolismo , Aparelho Lacrimal/citologia , Síndrome de Sjogren/metabolismo , Proteínas rab27 de Ligação ao GTP/metabolismo , Células Acinares/citologia , Células Acinares/metabolismo , Células Acinares/patologia , Animais , Carbacol/farmacologia , Células Cultivadas , Modelos Animais de Doenças , Endossomos/metabolismo , Aparelho Lacrimal/metabolismo , Aparelho Lacrimal/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Proteínas rab27 de Ligação ao GTP/genéticaRESUMO
OBJECTIVE: Dry eye syndrome in which tear fluid quality or abnormality, or kinetic abnormality is caused by various reasons, resulting in decreased tear film stability. In recent years, more and more results from the studies indicate that miRNA alterations are involved in dry eye syndrome. And miRNA-146a-5p is a key regulator to regulate the inflammatory response. In this paper, we demonstrated whether miRNA-146a-5p could cure dry eye syndrome by regulating target genes based on network analysis. METHODS: In current study, we collected the blood of patients with dry eye disease served as a model group; the blood of healthy people was served as control group. The expression of miRNA-146a-5p in the patients was detected by RT-PCR, the genes controlled by miRNA-146a-5p were predicted by TargetScan, miRDB, miRWalk, and PicTar databases, and the genes regulated by miRNA-146a-5p which relative with dry eye disease were selected by drawing Venn diagram. RESULTS: The comparison of the general information between patients and healthy people was no significant difference, and it indicated that the two groups were comparable. The results of databases showed that IRAK1 was one of the target genes regulated by miRNA-146a-5p, and it is related to dry eye disease. The expression of miRNA-146a-5p was negatively related to IRAK1 mRNA and protein, while IRAK1 had a positive correlation with IL-6, TNF-α, and CBP proteins. CONCLUSION: These results emphasized that miRNA-146a-5p could inhibit the expression of IRAK1, IL-6, TNF-α, and CBP to help reduce the inflammatory response in dry eye syndrome.
Assuntos
Síndromes do Olho Seco , MicroRNAs , Adulto , Animais , Estudos de Casos e Controles , Células Cultivadas , Biologia Computacional , Citocinas/sangue , Citocinas/metabolismo , Síndromes do Olho Seco/epidemiologia , Síndromes do Olho Seco/genética , Síndromes do Olho Seco/metabolismo , Feminino , Humanos , Inflamação/metabolismo , Quinases Associadas a Receptores de Interleucina-1/genética , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Aparelho Lacrimal/citologia , Masculino , MicroRNAs/sangue , MicroRNAs/genética , MicroRNAs/metabolismo , Pessoa de Meia-Idade , RatosRESUMO
Sjögren syndrome (SS) is an immunologically complex, chronic autoimmune disease targeting lacrimal and salivary glands. Nonobese diabetic (NOD) mice spontaneously develop inflammation of lacrimal and salivary glands with histopathological features similar to SS in humans including focal lymphocytic infiltrates in the affected glands. The innate immune signals driving lymphocytic infiltration of these glands are not well-defined. Here we evaluate the role of Toll-like receptor (TLR) 7 in the development of SS-like manifestations in NOD mice. We created a Tlr7 knockout NOD mouse strain and performed histological and gene expression studies to characterize the effects of TLR7 on autoimmunity development. TLR7 was required for male-specific lacrimal gland inflammation but not for female-specific salivary gland inflammation. Moreover, TLR7 was required for type 1 diabetes development in male but not female NOD mice. RNA sequencing demonstrated that TLR7 was associated with a type I interferon (IFN) response and a type I IFN-independent B cell response in the lacrimal glands. Together these studies identify a previously unappreciated pathogenic role for TLR7 in lacrimal gland autoimmunity and T1D development in male NOD mice adding to the growing body of evidence supporting sex differences in mechanisms of autoimmune disease in NOD mice.
Assuntos
Autoimunidade/genética , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Aparelho Lacrimal/imunologia , Glicoproteínas de Membrana/imunologia , Síndrome de Sjogren/imunologia , Receptor 7 Toll-Like/imunologia , Animais , Linfócitos B/imunologia , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica/imunologia , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Interferon Tipo I/metabolismo , Aparelho Lacrimal/citologia , Aparelho Lacrimal/patologia , Masculino , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , RNA-Seq , Glândulas Salivares/citologia , Glândulas Salivares/imunologia , Glândulas Salivares/metabolismo , Sexo , Receptor 7 Toll-Like/genéticaRESUMO
The lacrimal sac (LS) empties in the nasolacrimal duct to drain the tears in the inferior nasal meatus. Different studies indicated the role of the lacrimal pump in the lacrimal drainage. Although controversial, the lacrimal pump mechanism is an extrinsic one, either active, or passive. An intrinsic contractile potential of the LS was not documented previously. We thus aimed a retrospective immunohistochemical study to test the alpha-smooth muscle actin (α-SMA) and h-caldesmon expression in the LS wall. We used archived paraffin-embedded samples of LS from ten adult patients. The α-SMA + phenotype was detected in basal epithelial cells, in subepithelial ribbons of stromal cells, in vascular smooth muscle cells, as well as in pericytes. H-caldesmon was exclusively expressed in pericytes, vascular smooth muscle cells and myoepithelial cells of the subepithelial glands. The most striking feature we found in all samples was a consistent stromal network of α-SMA+/h-caldesmon- myofibroblasts. This finding supports an intrinsic scaffold useful for the lacrimal pump.
Assuntos
Aparelho Lacrimal/fisiologia , Miofibroblastos/fisiologia , Actinas/biossíntese , Proteínas de Ligação a Calmodulina/biossíntese , Células Epiteliais/metabolismo , Feminino , Humanos , Aparelho Lacrimal/anatomia & histologia , Aparelho Lacrimal/citologia , Masculino , Pessoa de Meia-Idade , Contração Muscular/fisiologia , Miócitos de Músculo Liso/metabolismo , Miofibroblastos/ultraestrutura , Pericitos/metabolismo , Estudos RetrospectivosRESUMO
Nasolacrimal duct obstruction (NLDO) is thought to be due to inflammation and fibrosis of lacrimal duct epithelial cells (LDECs). Here we investigated the effect of rebamipide, a drug that is used for the protection of the mucosa and the treatment of gastritis and gastroduodenal ulcers, on LDECs, both in vitro and in vivo. In this study, LDECs were cultured from rabbit lacrimal duct tissues, and the barrier function of LEDCs was examined in vitro via transepithelial electrical resistance (TER) measurement, with or without interleukin (IL)-6 and/or rebamipide. For the in vivo examination, benzalkonium chloride (BAC) was injected into the rabbit lacrimal ducts, followed by the application of rebamipide or a placebo vehicle alone. The results of the in vitro examination revealed a significant decrease in TER in the group treated with IL-6 alone compared with the placebo-vehicle group (p < 0.05) and the group treated with IL-6 and rebamipide (p < 0.01). The results of the in vivo examination revealed that the infiltration of neutrophils under the basement membrane and the disruption of tight junction proteins with BAC injection and rebamipide attenuates the disturbance of tissue construction. These results suggest that rebamipide protects LDECs via an anti-inflammatory effect and preserves the barrier function of those cells.
Assuntos
Alanina/análogos & derivados , Aparelho Lacrimal/efeitos dos fármacos , Quinolonas/farmacologia , Alanina/administração & dosagem , Alanina/farmacologia , Animais , Antiulcerosos/administração & dosagem , Antiulcerosos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Impedância Elétrica , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/fisiologia , Interleucina-6/administração & dosagem , Aparelho Lacrimal/citologia , Aparelho Lacrimal/fisiologia , Obstrução dos Ductos Lacrimais/patologia , Obstrução dos Ductos Lacrimais/fisiopatologia , Obstrução dos Ductos Lacrimais/prevenção & controle , Masculino , Microscopia Eletrônica de Varredura , Substâncias Protetoras/administração & dosagem , Substâncias Protetoras/farmacologia , Quinolonas/administração & dosagem , CoelhosRESUMO
BACKGROUND: Aqueous-deficient dry eye disease (ADDED) resulting from dysfunction of the lacrimal gland (LG) is currently incurable. Although LG stem/progenitor cell-based therapy is considered to be a promising strategy for ADDED patients, the lack of a reliable serum-free culture method to obtain enough lacrimal gland stem cells (LGSCs) and the basic standard of LGSC transplantation are obstacles for further research. METHODS: Adult mouse LGSCs were cultured in Matrigel-based 3D culture under serum-free culture condition, which contained EGF, FGF10, Wnt3A, and Y-27632. LGSCs were continuously passaged over 40 times every 7 days, and the morphology and cell numbers were recorded. LGSCs were induced to differentiate to ductal cells by reducing Matrigel rigidity, while fetal bovine serum was used for the induction of acinar cells. RT-PCR or qRT-PCR analysis, RNA-sequence analysis, H&E staining, and immunofluorescence were used for characterization and examining the differentiation of LGSCs. LGSCs were allotransplanted into diseased LGs to examine the ability of repairing the damage. The condition of eye orbits was recorded using a camera, the tear production was measured using phenol red-impregnated cotton threads, and the engraftments of LGSCs were examined by immunohistochemistry. RESULTS: We established an efficient 3D serum-free culture for adult mouse LGSCs, in which LGSCs could be continuously passaged for long-term expansion. LGSCs cultured from both the healthy and ADDED mouse LGs expressed stem/progenitor cell markers Krt14, Krt5, P63, and nestin, had the potential to differentiate into acinar or ductal-like cells in vitro and could engraft into diseased LGs and relieve symptoms of ADDED after orthotopic injection of LGSCs. CONCLUSION: We successfully established an efficient serum-free culture for adult mouse LGSCs aiming at LG repair for the first time. Our approach provides an excellent theoretical and technical reference for future clinical research for ADDED stem cell therapy.
Assuntos
Células Cultivadas/metabolismo , Aparelho Lacrimal/metabolismo , Células-Tronco/metabolismo , Animais , Humanos , Aparelho Lacrimal/citologia , Camundongos , Camundongos Endogâmicos NOD , Células-Tronco/citologiaRESUMO
The lacrimal gland (LG) is an exocrine tubuloacinar gland that secretes an aqueous layer of tear film. The LG epithelial tree is comprised of acinar, ductal epithelial, and myoepithelial cells (MECs). MECs express alpha smooth muscle actin (αSMA) and have a contractile function. They are found in multiple glandular organs and are of ectodermal origin. In addition, the LG contains SMA+ vascular smooth muscle cells of endodermal origin called pericytes: contractile cells that envelop the surface of vascular tubes. A new protocol allows us to isolate both MECs and pericytes from adult murine LGs and submandibular glands (SMGs). The protocol is based on the genetic labeling of MECs and pericytes using the SMACreErt2/+:Rosa26-TdTomatofl/fl mouse strain, followed by preparation of the LG single-cell suspension for fluorescence activated cell sorting (FACS). The protocol allows for the separation of these two cell populations of different origins based on the expression of the epithelial cell adhesion molecule (EpCAM) by MECs, whereas pericytes do not express EpCAM. Isolated cells could be used for cell cultivation or gene expression analysis.
Assuntos
Separação Celular/métodos , Células Epiteliais/citologia , Aparelho Lacrimal/citologia , Glândula Submandibular/citologia , Animais , Molécula de Adesão da Célula Epitelial/metabolismo , Regulação da Expressão Gênica , CamundongosRESUMO
In situ regeneration of lacrimal gland (LG) tissue would be a promising approach to curatively treat dry eye disease (DED). Mesenchymal stem cells (MSC) exhibit therapeutic effects in a variety of pathological conditions and our group recently reported that their number increases in regenerating mouse LG. Since the therapeutic effects are suggested to arise from secreted trophic factors, the application of MSC-secreted proteins seems to be a promising approach to induce/enhance LG regeneration. Therefore, this study aims to optimize the isolation of murine LG-MSC and analyze their secretome to investigate its potential for LG epithelial cell survival in vitro. For optimization, LG-MSC were isolated by an explant technique or cell sorting and their secretome was investigated under normal and inflammatory conditions. Results showed that the secretome of MSC had beneficial effects on the viability of ethanol-damaged LG epithelial cells. Additional, Lipocalin-2, prosaposin, ras GTPase-activating protein-binding protein 1 (Rac1) and signal transducer and activator of transcription 1 (STAT1), proteins that were up-regulated under inflammatory conditions, further improved the cell survival of ethanol-damaged LG epithelial cells. Interestingly, recovery of cell viability was highest, when the cells were incubated with STAT1. Summarizing, this study identified promising proteins for further studies on LG regeneration.
Assuntos
Células Epiteliais/metabolismo , Aparelho Lacrimal/metabolismo , Células-Tronco Mesenquimais/metabolismo , Animais , Sobrevivência Celular , Células Epiteliais/citologia , Aparelho Lacrimal/citologia , Masculino , Células-Tronco Mesenquimais/citologia , CamundongosRESUMO
Although single-cell RNA sequencing (scRNA-seq) has become one of the most powerful methods available for transcriptome analysis, the quality of scRNA-seq data largely depends on cell preparation. Cell preparation from cultured cells and tissues requires different methods because of the inherent differences between these two categories of cells. Compared to cultured cells, tissues have more extracellular matrix, and the cells are generally more adherent and thus difficult to dissociate. The challenge is to achieve sufficient dissociation, cell counts, and viability all at the same time. This protocol describes approaches that help achieve these goals. These include a cold dissociation technique using cryophilic proteases active at cold temperature, timing of trituration during protease digestion, as well as filtration and washing methods that optimize cell viability and retention. Materials and equipment that optimize the process also discussed. © 2019 by John Wiley & Sons, Inc.
Assuntos
Embrião de Mamíferos/citologia , Análise de Sequência de RNA/métodos , Animais , Separação Celular/métodos , Separação Celular/veterinária , Aparelho Lacrimal/citologia , Aparelho Lacrimal/embriologia , Camundongos , Glândulas Salivares/citologia , Glândulas Salivares/embriologia , Análise de Célula Única/métodosRESUMO
We present a protocol for isolation of putative epithelial progenitor cells from mouse lacrimal gland (LG) by fluorescence-activated cell sorting (FACS). Isolated LG epithelial progenitor cells can be cultured as 3D reaggregates within extracellular matrix gel or plated as a monolayer. 3D cultures could be maintained for several days and then dissociated with trypsin and plated as monolayer cultures, processed for analysis (e.g., mRNA/protein expression) and/or used for transplantations. Our goal is to provide researchers with a method that can be used as is or modified if isolation of other LG epithelial cell types is required.
Assuntos
Técnicas de Cultura de Células/métodos , Células Epiteliais/citologia , Aparelho Lacrimal/citologia , Células-Tronco/citologia , Animais , Células Cultivadas , Matriz Extracelular/metabolismo , Camundongos , Camundongos Endogâmicos C57BLRESUMO
PURPOSE: Primary fluid secretion in secretory epithelia relies on the unidirectional transport of ions and water across a single cell layer. This mechanism requires the asymmetric apico-basal distribution of ion transporters and intracellular Ca2+ signaling. The primary aim of the present study was to verify the localization and the identity of Ca2+-dependent ion channels in acinar cells of the mouse lacrimal gland. METHODS: Whole-cell patch-clamp-electrophysiology, spatially localized flash-photolysis of Ca2+ and temporally resolved digital Ca2+-imaging was combined. Immunostaining of enzymatically isolated mouse lacrimal acinar cells was performed. RESULTS: We show that the Ca2+-dependent K+-conductance is paxilline-sensitive, abundant in the luminal, but negligible in the basal membrane; and co-localizes with Cl--conductance. These data suggest that both Cl- and K+ are secreted into the lumen and thus they account for the high luminal [Cl-] (â¼141â¯mM), but not for the relatively low [K+] (<17â¯mM) of the primary fluid. Accordingly, these results also imply that K+ must be reabsorbed from the primary tear fluid by the acinar cells. We hypothesized that apically-localized Na+-K+ pumps are responsible for K+-reabsorption. To test this possibility, immunostaining of lacrimal acinar cells was performed using anti-Na+-K+ ATP-ase antibody. We found positive fluorescence signal not only in the basal, but in the apical membrane of acinar cells too. CONCLUSIONS: Based on these results we propose a new primary fluid-secretion model in the lacrimal gland, in which the paracellular pathway of Na+ secretion is supplemented by a transcellular pathway driven by apical Na+-K+ pumps.
Assuntos
Aparelho Lacrimal/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Alta/biossíntese , Lágrimas/metabolismo , Animais , Imuno-Histoquímica , Aparelho Lacrimal/citologia , Camundongos , Modelos Animais , Técnicas de Patch-ClampRESUMO
FGF signaling is a potent inducer of lacrimal gland development in the eye, capable of transforming the corneal epithelium into glandular tissues. Here, we show that genetic ablation of the Pea3 family of transcription factors not only disrupted the ductal elongation and branching of the lacrimal gland, but also biased the lacrimal gland epithelium toward an epidermal cell fate. Analysis of high-throughput gene expression and chromatin immunoprecipitation data revealed that the Pea3 genes directly control both the positive and negative feedback loops of FGF signaling. Importantly, Pea3 genes are also required to suppress aberrant Notch signaling which, if gone unchecked, can compromise lacrimal gland development by preventing the expression of both Sox and Six family genes. These results demonstrate that Pea3 genes are key FGF early response transcriptional factors, programing the genetic landscape for cell fate determination.
Assuntos
Diferenciação Celular/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Aparelho Lacrimal/crescimento & desenvolvimento , Fatores de Transcrição/metabolismo , Animais , Células Epidérmicas/fisiologia , Células Epiteliais/fisiologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Aparelho Lacrimal/citologia , Camundongos , Camundongos Knockout , Técnicas de Cultura de Órgãos , Receptores Notch/metabolismo , Fatores de Transcrição SOX/genética , Fatores de Transcrição SOX/metabolismo , Fatores de Transcrição/genéticaRESUMO
Severe dry eye syndrome resulting from lacrimal gland (LG) dysfunction can cause blindness, yet treatments remain palliative. In vitro reconstruction of LG tissue could provide a curative treatment. We aimed to combine epithelial cells with endothelial cells and mesenchymal stem cells (MSCs) to form a 3D functional unit. Epithelial cells and MSCs were isolated from porcine LG; endothelial cells were isolated from human foreskin. MSCs were characterised (flow cytometry and differentiation potential assays). All 3 cell types were combined on Matrigel and spheroid formation observed. Spheroids were characterised [immunohistochemistry (IHC) and transmission electron microscopy] and function assessed (ß-hexosaminidase assay). Spheroids were transferred to decellularised jejunum (SIS-Muc) in dynamic cultures for 1 week before further characterisation. MSCs did not express CD31 but expressed CD44 and CD105 and differentiated towards osteogenic and adipogenic lineages. Spheroids formed on Matrigel within 18 hr, contracting to ~10% of the well area (p < .005). IHC revealed presence of all 3 cells within spheroids. Transmission electron microscopy revealed cell-cell contacts and polarisation at the apical surface. In static cultures, function was increased in spheroids cf. monolayer controls (p < .05) but over 72 hr, spheroid function (p < .05), viability (p < .05), and proliferation decreased, whilst apoptosis increased. On SIS-Muc under dynamic culture, however, spheroids continued to proliferate to repopulate SIS-Muc. IHC revealed LG epithelial cells coexpressing pan-cytokeratin and lysozyme, as well as endothelial cells and MSCs and cells remained capable of responding to carbachol (p < .05). These spheroids could form the basis of a regenerative medicine treatment approach for dry eye syndrome. In vivo studies are required to evaluate this further.