Morphological observation of bone marrow mesenchymal stem cells under matrigel three dimensional culture conditions / Observación morfológica de células madre mesenquimales de médula ósea en condiciones de cultivo tridimensional con matrigel

SUMMARY: Matrigel is a basement membrane matrix extracted from the EHS mouse tumor containing extracellular matrix protein, its main components are laminin, type IV collagen, nestin, heparin sulfate, growth factor and matrix metalloproteinase.At room temperature, Matrigel polymerized to form a three dimensional matrix with biological activity. It can simulate the structure, composition, physical properties and functions of the cell basement membrane in vivo, which is beneficial to the culture and differentiation of the cells in vitro, and can be used for the study of cell morphology, biochemical function, migration, infection and gene expression. In this study, Matrigel three-dimensional culture model of bone marrow mesenchymal stem cells(BMSCs) was established, and its morphology, proliferation and survival were observed. BMSCs were isolated and cultured with whole bone marrow adherence method. The Second generation BMSCs with good growth condition were selected and mixed with Matrigel to form cell gel complexes. The morphology and proliferation of mesenchymal stem cells were observed by phase contrast microscope and HE staining,Live/Dead staining was used to evaluate the cell activity.Phase contrast microscopy showed that BMSCs were reticulated in Matrigel and proliferated well, After 7 days, the matrix gel gradually became soft and collapsed, a few cell reticular crosslinking growth was seen at 14 days; HE staining showed that the cytoplasm of the cells was larger on the fourth day and the cells were elongated and cross-linked on the seventh day; Live/dead staining showed that most cells showed green fluorescence with the prolongation of culture time, on the first, 4 and 7 days, the activity of bone marrow mesenchymal stem cells in Matrigel gradually increased, and the percentages were 92.57 %, 95.54 % and 97.37 %, respectively. Matrigel three-dimensional culture system can maintain the morphology, function and proliferation ability of bone marrow mesenchymal stem cells.

RESUMEN: Matrigel es una matriz de membrana basal extraída del tumor de ratón EHS que contiene proteína de matriz extracelular. Los componentes principales son laminina, el colágeno tipo IV, nestina, sulfato de heparina, factor de crecimiento y metaloproteinasa de matriz. A temperatura ambiente, Matrigel se polimerizó para formar una matriz tridimensional. Es posible simular la estructura, la composición, las propiedades físicas y las funciones de la membrana basal celular in vivo, lo que es beneficioso para el cultivo y la diferenciación de las células in vitro, y se puede utilizar para el estudio de la morfología celular, la función bioquímica, la migración, infección y expresión génica. En este estudio, se estableció el modelo de cultivo tridimensional Matrigel de células madre mesenquimales de médula ósea (BMSC), y se observó su morfología, proliferación y supervivencia. Las BMSC fueron aisladas y cultivadas con el método de adherencia de la médula ósea completa. Se seleccionaron las BMSC de segunda generación con buenas condiciones de crecimiento y se mezclaron con Matrigel para formar complejos de gel de células. La morfología y la proliferación de las células madre mesenquimales se observaron con microscopio de contraste de fase y se tiñó con Hematoxilina-Eosina (HE); para evaluar la actividad celular se usó la tinción Live/Dead. La microscopía de contraste mostró que las BMSC se reticularon en Matrigel y proliferaron bien. Después de 7 días, se observó que el gel de matriz gradualmente se volvió blando y colapsó, y se visualizó un cruce transversal de algunas células reticulares a los 14 días. La tinción mostró que la mayoría de las células mostraron una fluorescencia verde con la prolongación del tiempo de cultivo; en los primeros 4 y 7 días, la actividad de las células madre mesenquimales de la médula ósea en Matrigel aumentó gradualmente y los porcentajes fueron de 92,57 %, 95,54 % y 97,37 %, respectivamente. El sistema de cultivo tridimensional de Matrigel puede mantener la morfología, la función y la capacidad de proliferación de las células madre mesenquimales de la médula ósea.

Papel da laminina na migração de linfócitos T em modelo murino de transplante cardíaco alogênico / Role of laminina in the migration of T lymphocyte in a murine model of allogeneic cardiac transplantation

Durante o processo de rejeição, linfócitos T do receptor são ativados e migram para o enxerto. A glicoproteína laminina (LM) é importante na migração e posicionamento de linfócitos durante a rejeição, porém dados sobre o papel das diferentes isoformas neste processo são escassos. Usando um modelo de transplante cardíaco alogênico, nosso grupo verificou que o tratamento com o anticorpo monoclonal anti-LM diminuiu o infiltrado inflamatório e a deposição de LM nos enxertos cardíacos. No presente trabalho, caracterizamos os linfócitos T presentes nos linfonodos de drenagem e aloenxertos, a expressão das isoformas de LM nos aloenxertos e seu papel na migração dos linfócitos T. Nos aloenxertos, observamos um aumento de linfócitos T CD4+ e CD8+, e um maior enriquecimento de linfócitos CD8+ ativados, quando comparado aos enxertos isogênicos. Por qPCR, verificamos que somente a cadeia LAMB3, constituinte da LM332, teve sua expressão aumentada nos aloenxertos. Por imunofluorescência, verificamos uma maior deposição de LM332 e da cadeia LAMB3 nos aloenxertosPara verificar o papel da LM332 na migração dos linfócitos T, realizamos ensaios de migração e observamos uma migração reduzida dos linfócitos T frente a LM332, quando comparado aos grupos controle. Esse resultado sugere que a LM332 pode promover uma forte adesão, influenciando a migração dos linfócitos T alorreativos. A análise das isoformas de LM durante a rejeição será importante para o entendimento da migração das células efetoras durante esse processo e poderá ajudar a definir estratégias terapêuticas...

During rejection process, recipient T cells are activated in lymph nodes and migrate to the graft. The glycoprotein laminina (LM) is important in lymphocyte positioningand effector function during alloreactive responses. Using a model of allogeneic heart transplantation, where hearts from neonatal C57BL/6 mice were transplanted in the ear of adult BALB/c recipients, treatment with anti-LM mAb decreased the cellular infiltrate and LM deposition within the grafts. In this work, we characterized T cells inallografts, evaluate LM isoform expression and their role on T lymphocyte migration in the model described above. In allografts, we observed an increase of CD4+and CD8+ lymphocytes, and an enrichment of activated CD8+T cells, when compared tothe isografts. By qPCR, only LAMB3 chain, component of the LM332 isoform, was increased in allografts. Additionally, we observed higher deposition of LM332 in allografts by immunofluorescence. To check the role of LM332 on T cell migration, we performed ex vivo experiments and observed a reduced LM332-driven migratory response of lymphocytes from lymph nodes. Our data suggest that the predominanceof T cells in allografts can be related to an enhanced contact of T cells with LM332, as no haptotactic effect of the intact LM332 was observed. Analysis of the role of LM isoforms during the rejection process will be important to understand the trafficking process of effector cells during graft rejection and might help to define new therapeutical strategies...

Acivicin with glutaminase regulates proliferation and invasion of human MCF-7 and OAW-42 cells--an in vitro study.

Tumor cells intensely utilize glutamine as the major source of respiratory fuel. Glutamine-analogue acivicin inhibits tumor growth and tumor-induced angiogenesis in Ehrlich ascites carcinoma. In the present study, antitumor properties of acivicin in combination with glutaminase enzyme is reported. Acivicin along with E. coli glutaminase synergistically reduced in vitro proliferation and matrigel invasion of human MCF-7 and OAW-42 cells. Effects of single and combined treatments with acivicin and glutaminase on angiogenic factors were also analyzed in these cell lines. Co-administration of the treatment agents inhibits the release of VEGF and MMP-9 by cells in culture supernatant significantly than single agent treatments. The result suggests that combination of acivicin with glutaminase may provide a better therapeutic option than either of them given separately for treating human breast and ovarian cancer. However, further studies are required to be conducted in vivo for its confirmation.

Modulation of alpha1beta1 integrin mediated adhesion of hepatocytes to collagen IV and laminin by divalent cations.

Cell matrix interactions play a critical role in hepatic development and regeneration after acute injury. These interactions are mediated by transmembrane receptors belonging mainly to the integrin family. We have tried to assess the role of divalent cations in mediating attachment of hepatocytes to matrix proteins like collagen IV (Col IV) and laminin (Ln). The three cations examined viz. Ca2+, Mg2+ and Mn2+ showed attachment promoting activity. Since alpha1beta1 integrin is a common receptor for col IV and LN in liver, the effect of cations in its binding to these matrix proteins was studied. Although cations in general enhanced the binding, different cations exhibited differential effect in promoting the binding for different ligands. Mg2+ ions were more effective in promoting the binding of alpha1beta1 integrin to col IV but Ca2+ proved to be more effective one for Ln. Kinetic analysis of binding in dot blot assays using different concentrations of cations showed that while Mg2+ was active at low concentrations Ca2+ and Mn2+ promoted the binding more at higher concentrations. Absence of competitive effect in binding studies showed that they bind at different sites on the receptor. Differential effects of cations in promoting the binding of alpha1beta1 integrin to Col IV and Ln suggest that changes in level of diffusible cations can modulate affinity of the common receptor alpha1beta1 integrin to its ligands and can influence adhesion of hepatic cells to different matrix proteins during hepatic development and regeneration.

Galectinas: estructura, especificidad glicídica y ligandos específicos / Galectins: structure, sugar specificity and specific ligands

Las galectinas se definen por dos propiedades: secuencias de aminoácidos características compartidas y afinidad por azúcares ß-galactosídicos. Numerosas galactinas de mamíferos fueron secuenciadas y bien caracterizadas en diferentes especies, siendo clasificadas como galectina-1 a galectina-10, según sus homologías de secuencia. La identidad entre dominios que ligan carbohidratos de distintas galectinas de una especie de mamífero oscila entre 20-40 por ciento, mientras que la identidad de galectina-1, por ejemplo, entre distintas especies es de 80-90 por ciento. En la presente revisión, se describen las principales propiedades distintivas de las galectinas de mamífero en cuanto a estructura proteica, estructura cristalina, especificidad glicídica y ligandos específicos

Synthetic peptides related to laminin pentapeptide (YIGSR) fragment.

The synthetic laminin pentapeptide amide fragment (LF), Tyr-Ile-Gly-Ser-Arg-NH2 corresponding to a part of B1 chain of the glycoprotein, laminin, and six of its analogues having structural modifications at positions 1, 3 and 4 were synthesized by solid phase method employing mainly 9-fluorenylmethoxycarbonyl-amino acid trichlorophenyl esters as coupling agents and Merrifield resin as the solid support. Their biological activities were studied in vivo by lung tumor colonization assay and in vitro by cell adhesion assay. The activity of synthetic LF was found to correlate with the earlier reported results in both in vivo and in vitro assays. Among the analogues made, [Tyr4] LF and [Thr4]LF were found to inhibit the lung tumor colonies more efficiently than LF itself in the in vivo assay whereas [D- Ser4]LF exhibited almost the same inhibition as LF.