RESUMO
Although there have been many experimental studies demonstrating that bone marrow-derived mesenchymal stem cells (MSCs) have the potential to differentiate into mesenchymal tissues such as osteocytes, chondrocytes, and adipocytes in vivo and in vitro, little information is available regarding their potential to differentiate into lymphatic endothelial cells. Therefore, we chose to investigate differentiation of MSCs into lymphatic endothelial cells using stimulation with members of the vascular endothelial growth factor (VEGFs) family. Rat MSCs were isolated from bone marrow aspirate of Sprague-Dawley rats as previously described and characterized with flow cytometry for surface markers CD14, CD34, CD29, and CD90. Purified MSCs were plated and cultured in the presence of VEGF-A, VEGF-C, or the combination of both for 10 days. We examined the cells for Prox-1 and LYVE-1 by immunocytochemistry, RT-PCR, and Western blot analysis. Results demonstrated that compared to controls, cell differentiated with VEGF-A, VEGF-C and VEGF-A+VEGF-C expressed Prox-1 and LYVE-1. Our results indicate that MSCs induced by VEGFs are capable of differentiating into lymphatic endothelial-like cells in vitro, and this response has the potential to make them attractive candidates for the development of autologous tissue grafts for future therapy.
Assuntos
Biomarcadores/metabolismo , Medula Óssea/metabolismo , Diferenciação Celular , Células Endoteliais/citologia , Células-Tronco Mesenquimais/citologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Western Blotting , Medula Óssea/crescimento & desenvolvimento , Células Cultivadas , Células Endoteliais/metabolismo , Citometria de Fluxo , Técnicas Imunoenzimáticas , Masculino , Células-Tronco Mesenquimais/metabolismo , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Secondary lymphedema has been clinically well described, but a cure is still lacking. Although there have been previous investigations using plasmid DNA for gene therapy, few have focused on the use for the treatment of lymphedema. Therefore, we investigated the effects of VEGF-C gene transfer for the treatment of lymphedema using our plasmid pcDNA3.1-VEGF-C. We produced a surgical model of secondary lymphedema in the rat hindlimb and treated with local intradermal VEGF-C transfection to investigate the efficacy of gene transfer. Magnetic resonance imaging (MRI) (P<0.05), B ultrasound (P<0.05), and water displacement volumetry (P<0.05) demonstrated a reduction of lymphedema in therapy group as compared to controls. Histological and immunofluorescent studies demonstrated numerous newly formed lymphatic vessels in therapy group. Our results indicate that VEGF-C gene therapy has produced new lymphatic vessels which may have improved functional lymphatic drainage to reduce lymphedema volume in our model.
Assuntos
Terapia Genética/métodos , Linfedema/genética , Fator C de Crescimento do Endotélio Vascular/genética , Animais , DNA/genética , Técnicas de Transferência de Genes , Membro Posterior/irrigação sanguínea , Linfedema/diagnóstico por imagem , Linfedema/patologia , Imageamento por Ressonância Magnética , Neovascularização Fisiológica/genética , Plasmídeos/genética , Ratos , Ratos Wistar , Fluxo Sanguíneo Regional/fisiologia , UltrassonografiaRESUMO
BACKGROUND & OBJECTIVE: Vascular endothelial growth factor C (VEGF-C) discovered recently is a new member of vascular endothelial growth factor (VEGF) family. Studies have shown that VEGF-C is one specific regulating factor for lymphatic endothelial cells, and it expresses in a variety of human tumors. In order to explore the role of VEGF-C in the process of human tumor formation and development, the authors designed this study to examine the expressions of VEGF-C mRNA and protein in human breast cancer cell line MCF-7 and drug resistant cell line MCF-7/Adr. METHODS: According to the VEGF-C gene sequence, A specific oligonucleotide probe labeled with digoxin was designed and constructed; Then in situ hybridization was applied to detect the expression of VEGF-C mRNA in the cultured human breast cancer cell lines MCF-7 and MCF-7/Adr respectively; Finally immunohistochemistry was performed to examine the expression of VEGF-C protein in both cell lines. RESULTS: Blue positive staining granules for VEGF-C mRNA were observed in the cytoplasm of these cells by in situ hybridization where brown positive staining granules for VEGF-C protein were also observed by immunohistochemistry. While there were no positive staining granules in neither control cells. CONCLUSION: Human breast cancer cell lines MCF-7 and MCF-7/Adr can transcribe VEGF-C mRNA and translate corresponding protein in their cytoplasm.