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1.
Biol. Res ; 55: 30-30, 2022. ilus, tab, graf
Article in English | LILACS | ID: biblio-1403569

ABSTRACT

BACKGROUND: Xenotransplantation has been primarily performed using fresh donor tissue to study testicular development for about 20 years, and whether the cultured tissue would be a suitable donor is unclear. In this study, we combined testicular culture and xenotransplantation into an integrative model and explored whether immature testicular tissue would survive and continue to develop in this model. METHODS: In the new integrative model group, the testes of neonatal rats on postnatal day 8 (PND 8) were cultured for 4 days ex vivo and then were transplanted under the dorsal skin of castrated nude mice. The xenografted testes were resected on the 57th day after xenotransplantation and the testes of rats in the control group were harvested on PND 69. The survival state of testicular tissue was evaluated from morphological and functional perspectives including H&E staining, immunohistochemical staining of 8-OH-dG, immunofluorescence staining, TUNEL assay, ultrastructural study, gene expression and protein analysis. RESULTS: (a) We found that complete spermatogenesis was established in the testes in the new integrative model group. Compared with the control in the same stage, the seminiferous epithelium in some tubules was a bit thinner and there were vacuoles in part of the tubules. Immunofluorescence staining revealed some ACROSIN-positive spermatids were present in seminiferous tubule of xenografted testes. TUNEL detection showed apoptotic cells and most of them were germ cells in the new integrative model group. 8-OH-dG immunohistochemistry showed strongly positive-stained in the seminiferous epithelium after xenotransplantation in comparison with the control group; (b) Compared with the control group, the expressions of FOXA3, DAZL, GFRα1, BOLL, SYCP3, CDC25A, LDHC, CREM and MKI67 in the new integrative model group were significantly elevated (P < 0.05), indicating that the testicular tissue was in an active differentiated and proliferative state; (c) Antioxidant gene detection showed that the expression of Nrf2, Keap1, NQO1 and SOD1 in the new integrative model group was significantly higher than those in the control group (P < 0.05), and DNA methyltransferase gene detection showed that the expression of DNMT3B was significantly elevated as well (P < 0.05). CONCLUSION: The new integrative model could maintain the viability of immature testicular tissue and sustain the long-term survival in vivo with complete spermatogenesis. However, testicular genes expression was altered, vacuolation and thin seminiferous epithelium were still apparent in this model, manifesting that oxidative damage may contribute to the testicular development lesion and it needs further study in order to optimize this model.


Subject(s)
Animals , Male , Mice , Rats , Testis/metabolism , NF-E2-Related Factor 2/metabolism , Spermatogenesis , Acrosin/metabolism , Superoxide Dismutase-1/metabolism , Kelch-Like ECH-Associated Protein 1/metabolism , Methyltransferases/metabolism , Antioxidants/metabolism
2.
Journal of Chinese Physician ; (12): 1349-1353,1358, 2019.
Article in Chinese | WPRIM | ID: wpr-798098

ABSTRACT

Objective@#To explore potential therapeutic targets for neonatal hypoxic brain injury, we analyzed the effects of hypoxia on the gene expression profiles and signaling pathway in 3D cultured cerebral cortex cells.@*Methods@#R studio software was used to analyze the differentially expressed genes of hypoxia treated cerebral cortex cell data (GSE112137) which was downloaded from GEO database. Gene Oncology and KEGG software were used to enrich the molecular function, biological process and signaling pathways of differentially expressed genes. Then String and Cytoscape software were adapted to analyzed gene interaction network between these genes.@*Results@#There were 395 increasing genes and 185 decreasing genes (Change Fold≥2) were identified in hypoxic cerebral cells compared with the control groups. Most elevated genes were mainly related with molecular function including dioxygenase activity, isomerase activity and misfolded protein binding, while the decreasing genes were enriched in RNA polymerase Ⅱ proximal promoter sequence-specific DNA binding. Biological process enrichment analysis revealed that hypoxia up-regulated genes were associated with endoplasmic reticulum stress, oxidation-reduction process and glycolysis, while down-regulated genes were involved in the progress of neural development and cell differentiation. KEGG pathway enrichment results indicated hypoxia increasing genes were mainly related with endoplasmic reticulum protein processing, glycolysis, amino acid biosynthesis, and decreasing genes were mainly enriched in Parkinson′s disease signaling pathway.@*Conclusions@#Hypoxia in human cerebral cortex cells could cause endoplasmic reticulum stress, protein misfolding and metabolic abnormalities, inhibited the development of neuron cells. Drugs targeting these process may be beneficial to alleviate cerebral hypoxia injury.

3.
Journal of Chinese Physician ; (12): 1349-1353,1358, 2019.
Article in Chinese | WPRIM | ID: wpr-791148

ABSTRACT

Objective To explore potential therapeutic targets for neonatal hypoxic brain injury,we analyzed the effects of hypoxia on the gene expression profiles and signaling pathway in 3D cultured cerebral cortex cells.Methods R studio software was used to analyze the differentially expressed genes of hypoxia treated cerebral cortex cell data (GSE112137) which was downloaded from GEO database.Gene Oncology and KEGG software were used to enrich the molecular function,biological process and signaling pathways of differentially expressed genes.Then String and Cytoscape software were adapted to analyzed gene interaction network between these genes.Results There were 395 increasing genes and 185 decreasing genes (Change Fold ≥2) were identified in hypoxic cerebral cells compared with the control groups.Most elevated genes were mainly related with molecular function including dioxygenase activity,isomerase activity and misfolded protein binding,while the decreasing genes were enriched in RNA polymerase Ⅱ proximal promoter sequence-specific DNA binding.Biological process enrichment analysis revealed that hypoxia up-regulated genes were associated with endoplasmic reticulum stress,oxidation-reduction process and glycolysis,while down-regulated genes were involved in the progress of neural development and cell differentiation.KEGG pathway enrichment results indicated hypoxia increasing genes were mainly related with endoplasmic reticulum protein processing,glycolysis,amino acid biosynthesis,and decreasing genes were mainly enriched in Parkinson's disease signaling pathway.Conclusions Hypoxia in human cerebral cortex cells could cause endoplasmic reticulum stress,protein misfolding and metabolic abnormalities,inhibited the development of neuron cells.Drugs targeting these process may be beneficial to alleviate cerebral hypoxia injury.

4.
Chinese Journal of Dermatology ; (12): 138-142, 2018.
Article in Chinese | WPRIM | ID: wpr-710348

ABSTRACT

Objective To evaluate the effect of diammonium glycyrrhizinate extracted from the Chinese traditional medicine licorice root on the growth of human hair follicles cultured in vitro,and to detect the expression of wnt/β-catenin signaling pathway-related molecules.Methods Isolated hair follicles were cultured with diammonium glycyrrhizinate at different concentrations of 0.1,0.01,0.001 and 0.000 1 μmol/L for 10 days,and the hair follicles cultured in Williams' E medium without diammonium glycyrrhizinate served as a control group.The length of hair follicles was measured under a microscope every day,the morphologic changes of hair follicles were observed,and photos were taken.Immunofluorescence assay was performed to assess the proliferation of hair matrix cells,as well as to determine the expression of β-catenin,glycogen synthase kinase 3β (GSK3β),phosphorylated GSK3β (p-GSK3β) and lymphoid enhancer factor-1 (Lef1) in the Wnt/β-catenin signaling pathway.Statistical analysis was carried out by repeated-measures analysis of variance and one-way analysis of variance.Results As repeated-measures analysis of variance showed,only 0.01 μmol/L diammonium glycyrrhetate showed significantly promotive effect on the growth of hair follicles compared with the medium alone (P < 0.05),and there were no significant differences in the length of hair follicles between the other concentration groups and the control group.Compared with the control group,the transition to the catagen phase of human hair cycle was delayed in the 0.01-μmol/L diammonium glycyrrhetate group,while it did not change in the other diammonium glycyrrhetate groups and control group.Immunofluorescence assay showed that the number of ki67-positive hair matrix cells was obviously increased in the 0.1-,0.01-,0.001-μmol/L diammonium glycyrrhizinate groups compared with the control group,while there was no difference between the 0.000 1-μmol/L diammonium glycyrrhizinate group and the control group.One-way analysis of variance revealed that the expression of β-catenin,p-GSK3β and Lef1 significantly differed among all the groups (F =12.604,16.65,15.266 respectively,P < 0.05),while no significant difference in the expression of GSK3β was found among these groups (F =1.472,P > 0.05).Least significant difference (LSD)-t test revealed that the expre-ssion of β-catenin,p-GSK3β and Lef1 in the hair matrix cells was significantly higher in the 0.1-,0.01-,0.001-μmol/L diammonium glycyrrhizinate groups than in the control group (all P < 0.05),but there was no significant difference between the 0.000 1-μmol/L diammonium glycyrrhizinate group and the control group (P > 0.05).Conclusion Diammonium glycyrrhetate at the concentration of 0.01 μmol/L shows markedly promotive effect on the in vitro growth of hair follicles,and can increase the proliferative activity of hair matrix cells and delay the transition to the catagen phase,which may be associated with the activation of Wnt/β-catenin signaling pathway.

5.
Arq. gastroenterol ; 54(2): 130-134, Apr.-June 2017. tab, graf
Article in English | LILACS | ID: biblio-838836

ABSTRACT

ABSTRACT BACKGROUND The diarrheal syndrome is considered a serious public health problem all over the world and is considered a major cause of morbidity and mortality in developing countries. The high incidence of enteroaggregative Escherichia coli in diarrheal syndromes classified as an emerging pathogen of gastrointestinal infections. After decades of study, your pathogenesis remains uncertain and has been investigated mainly using in vitro models of adhesion in cellular lines. OBJECTIVE The present study investigated the interaction of enteroaggregative Escherichia coli strains isolated from childhood diarrhea with rabbit ileal and colonic mucosa ex vivo, using the in vitro organ culture model. METHODS The in vitro adhesion assays using cultured tissue were performed with the strains co-incubated with intestinal fragments of ileum and colon over a period of 6 hours. Each strain was tested with three intestinal fragments for each region. The fragments were analysed by scanning electron microscopy. RESULTS Through scanning electron microscopy we observed that all strains adhered to rabbit ileal and colonic mucosa, with the typical aggregative adherence pattern of “stacked bricks” on the epithelium. However, the highest degree of adherence was observed on colonic mucosa. Threadlike structures were found in greater numbers in the ileum compared to the colon. CONCLUSION These data showed that enteroaggregative Escherichia coli may have a high tropism for the human colon, which was ratified by the higher degree of adherence on the rabbit colonic mucosa. Finally, data indicated that in vitro organ culture of intestinal mucosa from rabbit may be used to elucidate the enteroaggregative Escherichia coli pathogenesis.


RESUMO CONTEXTO A síndrome diarréica é considerada um grave problema de saúde pública em todo o mundo e é considerada uma das principais causas de morbidade e mortalidade nos países em desenvolvimento. A elevada incidência de Escherichia coli enteroagregativa nas síndromes diarreicas a classificou como um patógeno emergente de infecções gastrintestinais. Depois de décadas de estudo, sua patogênese ainda é incerta e tem sido investigada usando principalmente modelos in vitro de adesão em linhagens celulares. OBJETIVO O presente estudo investigou a interação de cepas de Escherichia coli enteroagregativa isoladas de diarreia infantil com mucosa ileal e colônica de coelho ex vivo, utilizando o modelo de cultura de órgão in vitro. MÉTODOS Os ensaios de adesão in vitro utilizando tecido cultivado foram realizados com as cepas co-incubadas com fragmentos intestinais de íleo e de cólon durante um período de 6 horas. Cada cepa foi testada em três fragmentos intestinais para cada região. Os fragmentos foram analisados por microscopia eletrônica de varredura. RESULTADOS Através da microscopia eletrônica de varredura observamos que todas as cepas aderiram a mucosa ileal e colônica de coelho, com o padrão de aderência agregativo típico de “tijolos empilhados” no epitélio. Entretanto, o maior grau de adesão foi observado na mucosa do cólon. Estruturas filiformes foram encontradas em maior número no íleo em comparação com o cólon. CONCLUSÃO Esses dados mostraram que Escherichia coli enteroagregativa pode ter um maior tropismo para o cólon humano, o que foi ratificado pelo maior grau de aderência na mucosa do cólon de coelho. Finalmente, os dados indicaram que a cultura de órgão in vitro da mucosa intestinal de coelho pode ser utilizado para elucidar a patogênese de Escherichia coli enteroagregativa.


Subject(s)
Humans , Animals , Male , Bacterial Adhesion/physiology , Colon/microbiology , Escherichia coli/physiology , Escherichia coli Infections/microbiology , Ileum/microbiology , Intestinal Mucosa/microbiology , Phylogeny , Rabbits , Microscopy, Electron, Scanning , Colon/ultrastructure , Virulence Factors , Ileum/ultrastructure , Intestinal Mucosa/ultrastructure
6.
Int. j. morphol ; 35(2): 584-588, June 2017. ilus
Article in English | LILACS | ID: biblio-893025

ABSTRACT

Bone remodeling is a process regulated by the interaction between cells and various molecules such as parathyroid hormone (PTH). The aim of the study was to evaluate the effect of different doses of PTH on osteoclast activity in a culture model of bone organs. Six-day-old male C57BL/6 mice (n=14) were euthanized and the calvariae were dissected and sectioned in the middle, keeping the periosteal and endosteal. The bone fragments were divided into three groups: Group I (control - without adding PTH), Group II (addition of 3 nM PTH) and Group III (30 nM PTH), all cultured in aMEM for up to 72 h osteoclast activity was evaluated by biochemical quantification of calcium released in the culture medium at intervals of 24, 48, and 72 h and by histomorphometric analysis of bone resorption lacunae at 72 h our results show that group II exhibited significantly higher values of calcium levels in the medium compared to group I (p<0.05) in all intervals, also being higher for group III at 24 hours (p<0.05). Group II promoted a greater demineralization area (22068 ± 2193 mm2) than those found in group I (2084 ± 38 mm2) and group III (8952 ± 246 mm2), with statistically significant difference (p<0.001) among all groups. We concluded that in culture model of bone organs PTH promotes higher bone resorption when administered in lower doses.


La remodelación ósea es un proceso regulado por la interacción entre las células y varias moléculas como la hormona paratiroidea (PTH). El objetivo de este estudio fue evaluar el efecto de diferentes dosis de PTH sobre la actividad de los osteoclastos en un modelo de cultivo de órganos óseos. Se sacrificaron ratones C57BL/6 machos, de 6 días de edad (n = 14), y se disecaron y seccionaron las calvarias, manteniendo el periostio y endostio. Los fragmentos óseos se dividieron en tres grupos: Grupo I (control - sin adición de PTH), Grupo II (adición de 3 mM de PTH) y Grupo III (30 nM de PTH), todos cultivados en aMEM hasta 72 horas. La actividad de los osteoclastos se evaluó mediante la cuantificación bioquímica de calcio liberado en medio de cultivo, a intervalos de 24, 48 y 72 horas, y por análisis histomorfométrico de las lagunas de resorción ósea a las 72 horas. Nuestros resultados muestran que el grupo II exhibió valores significativamente más altos de calcio en el medio, comparado con el grupo I (p <0.05) en todos los intervalos, siendo también más alto para el grupo III a las 24 horas (p <0.05). El grupo II promovió una mayor área de desmineralización (22068 ± 2193 mm2) que los encontrados en el grupo I (2084 ± 38 mm2) y en el grupo III (8952 ± 246 mm2), con diferencia estadísticamente significativa (p <0,001) entre todos los grupos. Concluimos que en el modelo de cultivo de órganos óseos la PTH promueve una mayor resorción ósea cuando se administra en dosis más bajas.


Subject(s)
Animals , Male , Mice , Osteoclasts/drug effects , Osteoclasts/metabolism , Parathyroid Hormone/pharmacology , Bone Remodeling/drug effects , In Vitro Techniques , Mice, Inbred C57BL , Tissue Culture Techniques
7.
International Eye Science ; (12): 1060-1062, 2017.
Article in Chinese | WPRIM | ID: wpr-641223

ABSTRACT

Corneal endothelial cell(CEC)is the most critical part for the cornea, of which activity can influence the postoperative vision.It is very important for the clinical cornea preservation considering the function and its self-purification of donor cornea.There are a variety of classical methods, which can significantly prolong the saving time of donor cornea with its good quality of CEC.We reviewed the published papers about present preservation methods of cornea, which can give us many suggestions for the clinical cornea preservation.

8.
Clinical and Experimental Otorhinolaryngology ; : 112-118, 2014.
Article in English | WPRIM | ID: wpr-173821

ABSTRACT

OBJECTIVES: Glucocorticoids, such as dexamethasone (DEX), increase apoptosis in a variety of white cells in nasal polyps and apoptosis is an important factor in the resolution of inflammation. However, the mechanism of glucocorticoids induced apoptosis in nasal polyp remains unclear. In this study the authors evaluated which pathways were engaged in apoptosis induced by DEX in an ex vivo model of nasal polyps. METHODS: Nasal polyp tissues were cultured using an air-liquid interface method. Cultures were maintained in the absence or presence of DEX (10 or 100 microM) for 24 hours. To investigate the involvement of the apoptotic signaling pathways in nasal polyp, such as caspase cascades, Fas-FasL signaling pathway, mitochondrial pathway and p38 mitogen-activated protein kinase (MAPK)/JNK pathway, the authors performed reverse transcription-polymerase chain reaction and Western blotting. RESULTS: The expression ratios of FasL, activated form of caspase-8, caspase-9, and caspase-3 were significantly higher in DEX-treated polyps (P<0.01). In the Bcl-2 family expression, the anti-apoptotic molecules, Bcl-2 and Bcl-XL decreased, but pro-apoptotic molecules, Bax increased, and Bid and Bad were activated. In the conventional MAPKs, JNK, and the phospho-p38 MAPK were significantly higher, but phospho-extracellular signal-regulated kinase (ERK)1/2 was significantly lower in DEX-treated polyps (P<0.01). CONCLUSION: DEX induces apoptosis of nasal polyp via caspase cascades, Fas-FasL signaling pathway, mitochondrial pathway and p38 MAPK/JNK pathway.


Subject(s)
Humans , Apoptosis , Blotting, Western , Caspase 3 , Caspase 8 , Caspase 9 , Dexamethasone , Glucocorticoids , Inflammation , Nasal Polyps , Organ Culture Techniques , p38 Mitogen-Activated Protein Kinases , Phosphotransferases , Polyps , Protein Kinases
9.
Article in English | IMSEAR | ID: sea-140334

ABSTRACT

Background & objectives: Artificial corneal endothelium equivalents can not only be used as in vitro model for biomedical research including toxicological screening of drugs and investigation of pathological corneal endothelium conditions, but also as potential sources of grafts for corneal keratoplasty. This study was aimed to demonstrate the feasibility of constructing human corneal endothelium equivalents using human corneal endothelial cells and acellular porcine corneal matrix. Methods: Porcine corneas were decellularized with sodium dodecyl sulphate (SDS) solution. Human corneal endothelial cells B4G12 were cultured with leaching liquid extracted from the acellular porcine corneal matrix, and then cell proliferative ability was evaluated by MTT assay. B4G12 cells were transplanted to a rat corneal endothelial deficiency model to analyze their in vivo bio-safety and pump function, and then seeded and cultured on acellular porcine corneal matrix for two wk. Corneal endothelium equivalents were analyzed using HE staining, trypan blue and alizarin red S co-staining, immunofluorescence and corneal swelling assay. Results: The leaching liquid from acellular porcine corneal matrix had little influence on the proliferation ability of B4G12 cells. Animal transplantation of B4G12 cells showed that these cells had similar function to the native cells without causing a detectable immunological reaction and neoplasm in vivo. These formed a monolayer covering the surface of the acellular porcine corneal matrix. Trypan blue and alizarin red S co-staining showed that B4G12 cells were alive after two wk in organ culture and cell boundaries were clearly delineated. Proper localizations of ZO-1 and Na+/K+ ATPase were detected by immunofluorescence assay. Functional experiments were conducted to show that the Na+/K+ ATPase inhibitor ouabain could block the ionic-pumping function of this protein, leading to persistent swelling of 51.7 per cent as compared to the control. Interpretation & conclusions: Our findings showed that B4G12 cells served as a good model for native corneal endothelial cells in vivo. Corneal endothelium equivalents had properties similar to those of native corneal endothelium and could serve as a good model for in vitro study of human corneal endothelium.


Subject(s)
Biocompatible Materials/therapeutic use , Endothelium, Corneal , Endothelium, Corneal/transplantation , Humans , Organ Culture Techniques/methods , China
10.
Chinese Journal of Experimental Ophthalmology ; (12): 121-126, 2012.
Article in Chinese | WPRIM | ID: wpr-635792

ABSTRACT

BackgroundAllogenic immunological rejection is still the common cause of keratoplasty failure.Organ culture is a good choice for gradually attenuating corneal antigeniciy.However,the complicated regulatory mechanism of such allogenic rejection with organ culture gratts is unclear until now.ObjectiveTo investigate the changes of several Thl/Th2 cytokines and T lymphocyte subsets in the rat corneal allogenic transplantation rejection with organ culture grafts.MethodsThirty-six SPF Wistar rats served as donors,and 72 SPF SD rats served as recipients in this study.Seventy-two SD rats were divided into two groups randomly.Organ culture and fresh grafts from Wistar rats were performed on 36 recipients SD rats,respectively.Six normal SD rats were served as the normal control.After transplantation,graft survival time was observed ; Aqueous humor levels of IL-2,IFN-γ,IL-4 and TNF-α were measured by enzyme linked immunosorbent assay.Immunohistochemistry was performed to examine CD25 expressions in grafts.Levels of TNF-αmRNA,IFN-γmRNA,IL-4mRNA and CD25mRNA in grafts were detected by using reverse transcription polymerase chain reagction.The levels of CD28subsets in peripheralblood were determined by Flow Cytometry.The use of experimental animals followed the Statement of ARVO.Results The mean rat graft survival time was longer in the organ culture grafts group( 13.78 d) than that with fresh gratts( 10.56 d)(t=14.945,P =0.000 ).The aqueous humor levels of IL-2,IFN-γ,IL-4 and TNF-α were higher in two allograft groups at day 6,day 13 and day 24 after transplantation than that in the normal control,which peaked at day 13,and the fresh grafts group had the highest level( F=324.891,416.416,240.661,364.533,P=0.000).At day 13,CD25 was weakly expressed in frozen section of two operation groups.The expression of TNF-α and IFN-γmRNA in organ culture grafts group was markedly less than in fresh grafts group at day 13 after surgeries (t =2.464,P =0.039;t=5.438,P=0.001 ),whereas there was no significient difference in IL-4 and CD25 mRNA levels between them (t=-0.782,P =0.457,t =0.712,P =0.497).The percentages of CD28 of the fresh graft group increased significantly and more weakly expressed in organ culture grafts group (P =0.016 ). Conclusions Th1/Th2 cytokines and CD28 lymphocyte subsets may play important roles during corneal allograft rejection with organ culture grafts.

11.
Arq. gastroenterol ; 48(3): 199-204, July-Sept. 2011. ilus
Article in English | LILACS | ID: lil-599654

ABSTRACT

CONTEXT: Enteroaggregative Escherichia coli strains have been associated with persistent diarrhea in several developing countries. In vivo procedures with animal models, in vitro assays with cellular lines and in vitro organ culture with intestinal fragments have been utilized to study these bacteria and their pathogenicity. OBJECTIVE: The present experimental research assessed the pathogenic interactions of three enteroaggregative Escherichia coli strains, using the in vitro organ culture, in order to show the adherence to different regions of both, the ileal and the colonic mucosa and demonstrate possible mechanisms that could have the participation in the prolongation of diarrheiogenic process. METHODS: This study used intestinal fragments from terminal ileum and colon that were excised from pediatric patients undergoing intestinal surgeries and from adult patients that underwent to colonoscopic procedures. Each strain was tested with three intestinal fragments for each region. Tissue was fixed for scanning electron microscopic analysis. RESULTS: These bacteria colonized ileal and colonic mucosa in the typical stacked-brick configuration in the ileum and colon. In both regions, the strains were seen over a great amount of mucus and sometimes over the intact epithelium. In some regions, there is a probable evidence of effacement of the microvilli. It was possible to see adhered to the intestinal surface, bacteria fimbrial structures that could be responsible for the adherence process. CONCLUSION: In order to cause diarrhea, enteroaggregative Escherichia coli strains adhere to the intestinal mucosa, create a mucoid biofilm on the small bowel surface that could justify the digestive-absorptive abnormalities and consequently, prolonging the diarrhea.


CONTEXTO: Cepas de Escherichia coli enteroagregativa têm sido associadas à diarreia persistente em vários países em desenvolvimento. Procedimentos in vivo com modelos animais, cultura de órgão in vitro com fragmentos intestinais e ensaios in vitro com linhas celulares têm sido utilizados para estudar essas bactérias e a sua patogenicidade. OBJETIVO: A presente investigação experimental avaliou as interações patogênicas de três cepas de Escherichia coli enteroagregativa, usando cultura de órgão in vitro, para mostrar a aderência a diferentes regiões do intestino: íleo e cólons e demonstrar possíveis mecanismos que poderiam ter participação na perpetuação do processo diarréico. MÉTODOS: Este estudo usou fragmentos de íleo terminal e cólon que foram retirados de pacientes pediátricos submetidos a cirurgias intestinais e de pacientes adultos que foram submetidos a colonoscopias. Cada cepa foi testada com três fragmentos intestinais para cada região. O tecido foi fixado para análise sob microscopia eletrônica de varredura. RESULTADOS: Estas bactérias colonizaram mucosa ileal e colônica na configuração típica de pilhas de tijolos. Em ambas as regiões, as bactérias foram vistas sobre grande quantidade de muco e, às vezes, sobre o epitélio intacto. Em algumas áreas, há evidência de provável achatamento de vilosidades. Foi possível ver sobre a superfície intestinal, estruturas fimbriais bacterianas que poderiam estar relacionadas ao processo de adesão. CONCLUSÕES: Para causar diarreia, cepas de Escherichia coli enteroagregativa aderem à mucosa intestinal e criam um biofilme de muco sobre a superfície do intestino delgado, o que poderia justificar as anormalidades digestivo-absortivas e, por conseguinte, prolongar a diarreia.


Subject(s)
Adult , Child , Humans , Bacterial Adhesion , Colon/microbiology , Escherichia coli/pathogenicity , Ileum/microbiology , Intestinal Mucosa/microbiology , Colon/ultrastructure , Diarrhea/microbiology , Escherichia coli/ultrastructure , Ileum/ultrastructure , Intestinal Mucosa/ultrastructure , Microscopy, Electron, Scanning
12.
Korean Journal of Physical Anthropology ; : 105-112, 2011.
Article in English | WPRIM | ID: wpr-101460

ABSTRACT

Endochondral bone formation of the developing cranial base is a complex process. This mechanism requires precise orchestration of many cellular events and cartilage matrix metabolism, such as proliferation, becoming round in shape, termination of proliferation, hypertrophic size-increase, and finally programmed cell death. Active formation and degradation of cartilage matrix take place, in which microtubules are involved for intracellular events; bone apposition follows these events. However, the involvement of microtubules during these changes in the developing cranial base has not been identified yet. Thus, we investigated the involvement of microtubules in the regulation of endochondral bone formation during cranial base development. Using tubulin-binding drug nocodazole, we examined the effects of altering the structure and function of microtubules during in vivo organ culture of the mouse cranial base. Cultured specimens were analyzed with HE staining, immunohistochemistry, and cell counting in order to study the morphological and molecular changes that occurred in the tissues. Disruption of the microtubular array by nocodazole reduced cells expressing proliferation marker Ki67, osteogenic marker BSP, and BMP4 within the sphenooccipital synchondrosis region; chondrocyte hypertrophy was ceased in the hypertrophic zone; degeneration of cartilage matrix and bone matrix apposition was inhibited in the ossification center of the basooccipital cranial base. Our data demonstrated that disruption of microtubules by nocodazole have multiple inhibitory effects on the sequential changes that occur during endochondral bone formation, suggesting the importance of normal microtubule-polymerization in cranial base development.


Subject(s)
Animals , Mice , Bone Matrix , Bone Morphogenetic Protein 4 , Cartilage , Cell Count , Cell Death , Chondrocytes , Durapatite , Hypertrophy , Hypogonadism , Immunohistochemistry , Microtubules , Mitochondrial Diseases , Nocodazole , Ophthalmoplegia , Organ Culture Techniques , Osteogenesis , Skull Base
13.
Korean Journal of Dermatology ; : 373-379, 2010.
Article in Korean | WPRIM | ID: wpr-216995

ABSTRACT

BACKGROUND: Calcium plays a role in the proliferation and differentiation of keratinocytes. In a normal situation, the calcium concentration forms a gradient across the epidermal layers. Calcium is sparse in the basal layer and spinous layer. Skin organ culture is a useful model for conducting research on various aspects of skin biology. Skin organ culture systems are used for defining factors that affect homeostasis when elucidating the modulatory effects of biologic response modifiers, drugs and physical agents on the skin and also when studying complex aspects of cutaneous biology in normal and diseased skin. OBJECTIVE: In this study, we investigated the effects of extracellular calcium on the epidermis in a skin organ culture. METHODS: We compared the skin organ culture patterns under various culture conditions (calcium 0.1, 0.7, 1.4 and 2.0 mM). RESULTS: H&E staining showed different phenotypes according to the calcium concentration and IHC also showed different phenotyes compared to that of keratin 10, involucrin, filaggrin, loricrin and PCNA. CONCLUSION: As a result, we concluded that the calcium gradient is also an important factor in skin organ culture to maintain the vivo-like environment and the appropriate calcium concentration is 1.4 mM.


Subject(s)
Biology , Calcium , Epidermis , Homeostasis , Intermediate Filament Proteins , Keratin-10 , Keratinocytes , Membrane Proteins , Organ Culture Techniques , Phenotype , Proliferating Cell Nuclear Antigen , Protein Precursors , Skin
14.
Chinese Journal of Medical Aesthetics and Cosmetology ; (6): 402-404, 2009.
Article in Chinese | WPRIM | ID: wpr-380346

ABSTRACT

Objective To develop an in vitro model for cranial suture of neonatal SD rat. Methods The parietal hones with the sagittal suture were removed from SD rats (19-days old) for organ culture. In the experimental group, tensile force 3.92×10~(-3) N (0.4 g) was applied by helical springs, whereas no tension (0 N) was set in control group. These explants were observed under inverted microscope. At the end of the incubation period for 24 hours, general conditions were observed under inverted microscope and histological conditions were observed after hematoxylin and eosin stain. Results Under inverted micro-scope, sutures had no obvious changes in control group, whereas sutures were enlarged gradually in the experimental group. With histological observation, sutures developed normally in control group, but in experimental group, osteohlasts and capillary vessels proliferated actively in the suture. Conclusions In vitro model of cranial suture can be cultured and grown successively.

15.
Clinics ; 63(5): 683-688, 2008. ilus, graf
Article in English | LILACS | ID: lil-495045

ABSTRACT

INTRODUCTION: Saphenous vein grafting is still widely used to revascularize ischemic myocardium. The effectiveness of this procedure is limited by neointima formation and accelerated atherosclerosis, which frequently leads to graft occlusion. A better understanding of this process is important to clarify the mechanisms of vein graft disease and to aid in the formulation of strategies for prevention and/or therapeutics. OBJECTIVE: To develop an ex vivo flow system that allows for controlled hemodynamics in order to mimic arterial and venous conditions. METHODS: Human saphenous veins were cultured either under venous (flow: 5 ml/min) or arterial hemodynamic conditions (flow: 50 ml/min, pressure: 80 mmHg) for 1-, 2- and 4-day periods. Cell viability, cell density and apoptosis were compared before and after these intervals using MTT, Hoeschst 33258 stain, and TUNEL assays, respectively. RESULTS: Fresh excised tissue segments were well preserved prior to the study. Hoechst 33258 and MTT stains showed progressive losses in cell density and cell viability in veins cultured under arterial hemodynamic conditions from 1 to 4 days, while no alterations were observed in veins cultured under venous conditions. Although the cell density from 1-day cultured veins under arterial conditions was similar to that of freshly excised veins, the TUNEL assay indicated that most of these cells were undergoing apoptosis. CONCLUSION: The results observed resemble the events taking place during early in vivo arterial-vein grafting and provide evidence that an ex vivo perfusion system may be useful for the identification of new therapeutic targets that ameliorate vein graft remodeling and increase graft patency over time.


Subject(s)
Humans , Hemodynamics , Models, Cardiovascular , Organ Culture Techniques/methods , Perfusion/methods , Saphenous Vein/pathology , Analysis of Variance , Apoptosis/physiology , Cell Count , Cell Survival/physiology , In Situ Nick-End Labeling , Staining and Labeling , Saphenous Vein/transplantation , Saphenous Vein/ultrastructure
16.
The Journal of the Korean Orthopaedic Association ; : 721-729, 2006.
Article in Korean | WPRIM | ID: wpr-652854

ABSTRACT

PURPOSE: To investigate the potential of nucleus pulposus (NP) regeneration with a nanofiber-based scaffold inserted in the degenerative disc. MATERIALS AND METHODS: A nanofiber scaffold was fabricated using an electrospinning technique. In the in-vitro study, a total 18 discs with endplates on both sides were obtained from six New Zealand White (NZW) rabbits. A small volume of NP was removed through the hole from the endplate. The specimens were classified into three groups, intact (normal), inserting nanofiber scaffold (nanofiber), and defect (defect) group. The discs were analyzed by MRI scan and histological analysis. Six NZW rabbits were used in the in-vivo study. An annulotomy was performed through the dorsal approach L2-3 and L3-4 disc. A nanofibrous sheet type scaffold was inserted at L3-4. X-ray, MRI and histology analysis were carried out at 4, 8, 12 weeks after surgery. RESULTS: In the in-vitro study, the Nanofiber groups showed higher signal intensity and cell proliferation than the defect groups. In the in-vivo study, the Nanofiber and defect groups showed significant degeneration but there was no significant difference between these groups. CONCLUSION: Nanofiber scaffold might provide a favorable environment for regenerating disc cells. However, a defect in the annulus fibrosus (AF) might delay the regeneration of the disc cell at the nanofiber group. Therefore, NP regeneration using any scaffold should be examined along with AF regeneration for effective clinical applications.


Subject(s)
Animals , Rabbits , Cell Proliferation , Intervertebral Disc , Magnetic Resonance Imaging , Nanofibers , New Zealand , Organ Culture Techniques , Regeneration
17.
Basic & Clinical Medicine ; (12)2006.
Article in Chinese | WPRIM | ID: wpr-589257

ABSTRACT

Objective To verify the application of anterior segment perfusion culture and trabecular meshwork (TM) organ culture for glaucoma study. Methods TM tissue was cultured by perfused anterior segment and TM organ culture, light microscopy was used to observe the TM cells and intercellular spaces.Results IOP of the porcine anterior segments perfused under constant flow at 0.1 mL/h could bekept in normal range(10~12 mmHg). The IOP was elevated with the increasing of perfusion rate, while the morphology and structure of the tissue were well preserved. TM cultured by TM organ culture could also reserve the tissues well, but the intercelluar spaces collapsed. Conclusion Anterior segment perfusion model could be a short-term high-pressure model and may simulate the normal physical state. Adequate perfusion was necessary for normal TM.

18.
Korean Journal of Dermatology ; : 1178-1185, 2006.
Article in Korean | WPRIM | ID: wpr-185925

ABSTRACT

BACKGROUND: Hair loss including androgenetic alopecia and chronic telogen effluvium is recognized increasingly as a physically and psychologically harmful medical condition. Mesotherapy is considered as a new therapeutic modality for hair loss. OBJECTIVE: We studied to determine the effect of medications used in mesotherapy on hair organ culture and culture of dermal papilla cells. METHODS: First, occipital hair follicles were collected from patients with androgentic alopecia and separated into single hair follicles. The single hair follicles were cultured in William E media mixed with mesotherapy medications such as lidocaine, placental extract, Pondil(R), CRP-1000(R), and mixture of all these medications at different concentrations (1, 10, 50 microliter). On the 8th day, the cultured single hairs were stained with H&E and the length of those was measured under a microscope to compare with control group. Immunofluorescent study was performed to check expression of Ki-67, Bcl-2 and Bax on the hairs. Second, dermal papilla cells were isolated from occipital anagen hairs of patients with androgenetic alopecia and cultured in Dulbeco's modified Eagle's medium (DMEM). The mesotherapy medicines were added to the medium with one and two thousand dermal papilla cells, respectively. At the 3rd day, survival of the cells was evaluated with ELISA method comparing with control group. RESULTS: There were no statistical differences of the length of the hairs and the survival of the dermal papilla cells between experimental and control groups. With Bcl-2, we couldn't see any differences between experimental and control groups. With Ki-67, experimental groups showed less expression than control group. On the contrary, experimental groups showed more expression than control group in case of Bax. CONCLUSION: We can conclude from the results that the four medications used in mesotherapy are not effective for growth of cultured hair follicles and survival of cultured dermal papilla cells. However, more study would be needed for the establishment of objective and scientific evidences supporting mesotherapy and we should be in search for new medications for mesotherapy.


Subject(s)
Humans , Alopecia , Enzyme-Linked Immunosorbent Assay , Hair Follicle , Hair , Lidocaine , Mesotherapy , Organ Culture Techniques
19.
Yonsei Medical Journal ; : 249-254, 2006.
Article in English | WPRIM | ID: wpr-51471

ABSTRACT

The aim of the present study was to examine the functional changes that occur when a rabbit carotid artery is cultured in serum-free medium. In endothelium (EC)-intact arteries cultured under serum-free conditions, acetylcholine (ACh)-induced relaxation responses were partially, yet significantly, reduced when compared with freshly isolated arteries. After pretreatment with N(G)-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor, application of ACh resulted in a significant contraction in organ cultured arteries. The amplitude of the ACh-induced contractions increased with the duration of culture. In EC-denuded arteries cultured under serum-free conditions, ACh induced responses similar to those in EC-intact arteries pretreated with L-NAME. Furthermore, ACh caused a significant increase in intracellular Ca2+ concentration ([Ca2+]i) in EC-denuded arteries cultured under serum-free condition for 7 days. There was little change in either [Ca2+]i or tension in freshly isolated carotid rings. There was no difference in sodium nitroprusside-induced relaxation responses between fresh and cultured arteries. These results suggest that prolonged culture of carotid arteries under serum-free conditions changes the functional properties of vascular reactivity in rabbit carotid arteries.


Subject(s)
Rabbits , Animals , Time Factors , Organ Culture Techniques/methods , Nitroprusside/pharmacology , NG-Nitroarginine Methyl Ester/metabolism , Muscle Contraction , Models, Statistical , Dose-Response Relationship, Drug , Culture Media, Serum-Free/metabolism , Carotid Arteries/drug effects , Calcium/metabolism , Acetylcholine/pharmacology
20.
Korean Journal of Dermatology ; : 450-454, 2005.
Article in Korean | WPRIM | ID: wpr-169837

ABSTRACT

BACKGROUND: Skin organ culture is widely used as a tool to investigate skin biology or skin disease. OBJECTIVE: The objective of the present study was to develop an ideal skin organ culture model for evaluation of melanin pigmentation. METHODS: An air-liquid interface and submerged method were used. The histology of the cultured skin was studied with H&E stain. To examine the epidermal pigmentation, Fontana-Masson stain and NKI/beteb stain were performed. Pigment modifiers (arbutin, LY294002) were applied to the culture medium for 3 days as an air-liquid interface culture. RESULTS: The general architecture of the skin was well maintained for 5 days. The melanin pigment decreased during culture without change of the number of melanocytes. As expected from previous reports, the effect of pigment modifiers (arbutin, LY294002) on cultured skin was demonstrated. CONCLUSION: The results indicate that this skin organ culture model is useful in evaluating the melanin pigmentation


Subject(s)
Biology , Melanins , Melanocytes , Organ Culture Techniques , Pigmentation , Skin Diseases , Skin
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