Engenharia de vetores expressando o receptor de TGFβ1 tipo II solúvel sob controle do promotor NF-kB e análise da expressão em culturas musculares 2D e 3D
Data
2023-07-24
Autores
Sales, Sofia Mila Cantu 
Tipo
Dissertação de mestrado
Título da Revista
ISSN da Revista
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Resumo
Introdução: O TGF-β1 (Transforming growth factor beta 1) é um fator importante na cicatrização e formação de fibrose, cuja inibição é terapeuticamente relevante em lesões com perda muscular significativa. No presente trabalho, foi avaliada a funcionalidade do vetor lentiviral carreando o receptor solúvel de TGFβ (sTGFβRII) juntamente com o promotor induzível pelo NF-kB (fator de transcrição nuclear ligado na cadeia leve kappa das células B), que é altamente expresso durante a fase inflamatória da lesão muscular, em modelos de lesão 2D e 3D de células musculares. Objetivo: Desenvolvimento de vetores expressando o receptor de TGF-β1 tipo II solúvel sob o controle do promotor NF-kB e a avaliação da expressão em culturas musculares 2D e 3D. Metodologia: Células C2C12 foram transduzidas com vetores 1 (NFkb_Luciferase) ou 3 (NFkB_sTGβFRII). Essas linhagens produzidas foram cultivadas com meio DMEMc (Dulbecco’s modified Eagle’s medium – DMEM; 10% de soro fetal bovino (SFB); 1% Penicilina/estreptomicina (P/S)). Os esferoides de MDSC (muscle derived-stem cells) e C2C12 foram produzidos em micromoldes de agarose e cultivados com os meios de proliferação (PM) (Dulbecco’s modified Eagle’s medium – DMEM; 10% de soro fetal bovino (SFB); 10% de soro de cavalo (SC); 1% Penicilina/estreptomicina (P/S); 0,5% de extrato de embrião de galinha) e com o meio de diferenciação (DM) (DMEM; 10% SFB; 1% P/S). Foram produzidos também esferoides das linhagens 1 e 3. A caracterização dos esferoides foi realizada por diversos ensaios: viabilidade por citometria de fluxo
(7AAD e Anexina V), CellTiter-Glo, live and dead (calceína e 7AAD) e MTT. Histologia em historresina com coloração de azul de toluidina, imuno-histoquímica e imunofluorescência para os marcadores MyoD, Pax7 e eMyHC e a criação do modelo de lesão com cloreto de bário (BaCl2) e cardiotoxina (CTX). Resultados: Nos ensaios 2D, observamos uma modulação da expressão gênica do sTGFβRII e luciferase na presença de BaCl2. Caracterizamos esferoides de MDSC e C2C12 com forma e tamanho homogêneos tanto no meio padrão de cultivo quanto no meio de DM. A viabilidade dos esferoides do grupo PM foi alta (88% no 2º dia e 74% no 14º dia), enquanto a viabilidade do grupo DM diminuiu para 44%, e a porcentagem de células apoptóticas aumentou para 56% no 14º dia. Na análise da morfologia,
observamos um aumento de células fusiformes, especialmente no grupo DM, sugerindo a diferenciação. Também houve um aumento significativo na intensidade de fluorescência de eMyHC (7,5X), Pax-7 (2,1X) e MyoD (2X) em relação ao grupo PM no dia 14. O grupo PM mostrou uma capacidade de proliferação 5X maior que o grupo DM após o 4º dia, tanto em número total de células quanto nas células aderidas à placa. Os esferoides cultivados em meio PM também apresentaram uma maior capacidade de proliferação e fusão quando comparados ao meio DM. Nos esferoides C2C12, observamos um crescimento exponencial com um platô a partir do dia 14 em relação ao volume, e uma diminuição do volume do centro necrótico a partir do 4º dia. Na padronização do modelo de lesão por CTX e BaCl2,
observamos uma redução na viabilidade dos esferoides em todos os grupos quando comparados ao controle (CTL) (p<0,05), exceto no grupo BaCl2 6,25%. As concentrações de BaCl2 de 50%, 12,5% e 6,25%, e CTX (1µM) apresentaram viabilidades de 27%, 53%, 74% e 24%, respectivamente. A lesão induzida com BaCl2 50% levou a uma redução de 60,5% do volume do esferoide (p<0,05) e 54% da viabilidade (p<0,05) em comparação com o grupo controle. Esferoides das linhagens 1 e 3 mostraram uma redução do tamanho dos esferoides da linhagem 3 (p<0,05), sugerindo a importância do TGF-β1 para a proliferação celular e a possibilidade de sua depleção do meio de cultura. Também observamos uma redução na viabilidade dos esferoides que superexpressaram o sTGFβRII (p<0,05). No modelo transwell, verificamos que o receptor solúvel foi expresso e teve um efeito nas células C2C12, reduzindo sua viabilidade frente à lesão com BaCl2. Conclusão: foi possível produzir e caracterizar esferoides de células musculares usando a técnica de micromoldes de agarose scaffold-free. Observamos que as células MDSC foram capazes de se diferenciar ao longo do tempo, apresentando maior expressão de marcadores miogênicos no meio DM. Além disso, desenvolvemos um modelo de lesão em esferoides de células C2C12 utilizando BaCl2 e CTX, o qual resultou em redução da viabilidade e alteração no volume dos esferoides. Demonstramos que o receptor solúvel foi expresso e modulado por BaCl2, no entanto, sua expressão levou a uma diminuição no tamanho e na viabilidade dos esferoides. Embora sejam necessários estudos adicionais para compreender o efeito do receptor solúvel na redução da fibrose, nosso estudo evidenciou sua funcionalidade e modulação na presença de agentes indutores de lesão muscular, como o BaCl2.
Introduction: The TGF-β1 (Transforming growth factor beta 1) is an important factor in wound healing and fibrosis formation, and its inhibition is therapeutically relevant in injuries with significant muscle loss. In the present work, we evaluated the functionality of the lentiviral vector carrying the soluble TGFβ receptor (sTGFβRII) together with the promoter inducible by NF-kB (nuclear transcription factor linked to the kappa light chain of B cells), which is highly expressed during inflammatory phase of muscle injury, in 2D and 3D muscle cell injury models. Objective: Development of vectors expressing soluble TGF-β1 type II receptor under the control of the NF-kB promoter and evaluation of expression in 2D and 3D muscle cultures. Methodology: C2C12 cells were transduced with vectors 1 (NFkb_Luciferase) or 3 (NFkB_sTGβFRII). These produced lines were cultivated with DMEMc medium (Dulbecco's modified Eagle's medium - DMEM; 10% Fetal Bovine Serum (FBS); 1% Penicillin/Streptomycin (P/S)). MDSC (muscle derived stem cells) and C2C12 spheroids were produced in agarose micromolds and cultured with proliferation media (PM) (Dulbecco's modified Eagle's medium – DMEM; 10% fetal bovine serum (SFB); 10% horse serum (SC); 1% Penicillin/streptomycin (P/S); 0.5% chicken embryo extract) and with the differentiation medium (DM) (DMEM; 10% FBS; 1% P/S ). Lineages 1 and 3 spheroids were also produced. The spheroids were characterized using various assays: flow cytometry viability assay (7AAD and Annexin V), CellTiter-Glo assay, live and dead assay (calcein and 7AAD), and MTT assay. Histology with toluidine blue staining, immunohistochemistry, and immunofluorescence for the markers MyoD, Pax7, and eMyHC were performed, and a muscle injury model was created using barium chloride (BaCl2) and cardiotoxin (CTX). Results: In the 2D assays, we observed modulation of sTGFβRII gene expression and luciferase in the presence of BaCl2. We characterized MDSC and C2C12 spheroids with homogeneous shape and size in both standard culture medium and DM. The viability of PM group spheroids was high (88% on day 2 and 74% on day 14), while the viability of the DM group decreased to 44%, and the percentage of apoptotic cells increased to 56% on day 14. Morphological analysis revealed an increase in fusiform cells, especially in the DM group, suggesting differentiation. There was also a significant increase in the fluorescence intensity of eMyHC (7.5X), Pax-7 (2.1X), and MyoD (2X) compared to the PM group on day 14. The PM group exhibited 5X higher proliferation capacity than the DM group after day 4, both in total cell number and cells adhered to the plate. Spheroids cultured in PM medium also showed greater proliferation and fusion capacity compared to DM medium. In C2C12 spheroids, we observed exponential growth with a plateau from day 14 in terms of volume, and a decrease in the volume of the necrotic center from day 4. In the CTX and BaCl2 injury model standardization, we observed a reduction in spheroid viability in all groups compared to the control (CTL) (p<0.05), except for the BaCl2 6.25% group. The concentrations of 50%, 12.5%, and 6.25% BaCl2, and CTX (1µM) showed viabilities of 27%, 53%, 74%, and 24%, respectively. Injury induced by 50% BaCl2 led to a 60.5% reduction in spheroid volume (p<0.05) and 54% reduction in viability (p<0.05) compared to the control group. Spheroids from lineages 1 and 3 showed a reduction in size for lineage 3 spheroids (p<0.05), suggesting the importance of TGF-β1 for cell proliferation and the possibility of its depletion from the culture medium. We also observed a reduction in viability of spheroids that overexpressed sTGFβRII (p<0.05). In the transwell model, we found that the soluble receptor was expressed and had an effect on C2C12 cells, reducing their viability in response to BaCl2-induced injury. Conclusion: It was possible to produce and characterize muscle cell spheroids using the scaffold-free agarose micromold technique. We observed that MDSC cells were able to differentiate over time, showing higher expression of myogenic markers in DM medium. Additionally, we developed a model of injury in C2C12 cell spheroids using BaCl2 and CTX, which resulted in reduced viability and alterations in spheroid volume. We demonstrated that the soluble receptor was expressed and modulated by BaCl2; however, its expression led to a decrease in spheroid size and viability. Although further studies are needed to understand the effect of the soluble receptor in reducing fibrosis, our study provided evidence of its functionality and modulation in the presence of muscle injury-inducing agents like BaCl2.
Introduction: The TGF-β1 (Transforming growth factor beta 1) is an important factor in wound healing and fibrosis formation, and its inhibition is therapeutically relevant in injuries with significant muscle loss. In the present work, we evaluated the functionality of the lentiviral vector carrying the soluble TGFβ receptor (sTGFβRII) together with the promoter inducible by NF-kB (nuclear transcription factor linked to the kappa light chain of B cells), which is highly expressed during inflammatory phase of muscle injury, in 2D and 3D muscle cell injury models. Objective: Development of vectors expressing soluble TGF-β1 type II receptor under the control of the NF-kB promoter and evaluation of expression in 2D and 3D muscle cultures. Methodology: C2C12 cells were transduced with vectors 1 (NFkb_Luciferase) or 3 (NFkB_sTGβFRII). These produced lines were cultivated with DMEMc medium (Dulbecco's modified Eagle's medium - DMEM; 10% Fetal Bovine Serum (FBS); 1% Penicillin/Streptomycin (P/S)). MDSC (muscle derived stem cells) and C2C12 spheroids were produced in agarose micromolds and cultured with proliferation media (PM) (Dulbecco's modified Eagle's medium – DMEM; 10% fetal bovine serum (SFB); 10% horse serum (SC); 1% Penicillin/streptomycin (P/S); 0.5% chicken embryo extract) and with the differentiation medium (DM) (DMEM; 10% FBS; 1% P/S ). Lineages 1 and 3 spheroids were also produced. The spheroids were characterized using various assays: flow cytometry viability assay (7AAD and Annexin V), CellTiter-Glo assay, live and dead assay (calcein and 7AAD), and MTT assay. Histology with toluidine blue staining, immunohistochemistry, and immunofluorescence for the markers MyoD, Pax7, and eMyHC were performed, and a muscle injury model was created using barium chloride (BaCl2) and cardiotoxin (CTX). Results: In the 2D assays, we observed modulation of sTGFβRII gene expression and luciferase in the presence of BaCl2. We characterized MDSC and C2C12 spheroids with homogeneous shape and size in both standard culture medium and DM. The viability of PM group spheroids was high (88% on day 2 and 74% on day 14), while the viability of the DM group decreased to 44%, and the percentage of apoptotic cells increased to 56% on day 14. Morphological analysis revealed an increase in fusiform cells, especially in the DM group, suggesting differentiation. There was also a significant increase in the fluorescence intensity of eMyHC (7.5X), Pax-7 (2.1X), and MyoD (2X) compared to the PM group on day 14. The PM group exhibited 5X higher proliferation capacity than the DM group after day 4, both in total cell number and cells adhered to the plate. Spheroids cultured in PM medium also showed greater proliferation and fusion capacity compared to DM medium. In C2C12 spheroids, we observed exponential growth with a plateau from day 14 in terms of volume, and a decrease in the volume of the necrotic center from day 4. In the CTX and BaCl2 injury model standardization, we observed a reduction in spheroid viability in all groups compared to the control (CTL) (p<0.05), except for the BaCl2 6.25% group. The concentrations of 50%, 12.5%, and 6.25% BaCl2, and CTX (1µM) showed viabilities of 27%, 53%, 74%, and 24%, respectively. Injury induced by 50% BaCl2 led to a 60.5% reduction in spheroid volume (p<0.05) and 54% reduction in viability (p<0.05) compared to the control group. Spheroids from lineages 1 and 3 showed a reduction in size for lineage 3 spheroids (p<0.05), suggesting the importance of TGF-β1 for cell proliferation and the possibility of its depletion from the culture medium. We also observed a reduction in viability of spheroids that overexpressed sTGFβRII (p<0.05). In the transwell model, we found that the soluble receptor was expressed and had an effect on C2C12 cells, reducing their viability in response to BaCl2-induced injury. Conclusion: It was possible to produce and characterize muscle cell spheroids using the scaffold-free agarose micromold technique. We observed that MDSC cells were able to differentiate over time, showing higher expression of myogenic markers in DM medium. Additionally, we developed a model of injury in C2C12 cell spheroids using BaCl2 and CTX, which resulted in reduced viability and alterations in spheroid volume. We demonstrated that the soluble receptor was expressed and modulated by BaCl2; however, its expression led to a decrease in spheroid size and viability. Although further studies are needed to understand the effect of the soluble receptor in reducing fibrosis, our study provided evidence of its functionality and modulation in the presence of muscle injury-inducing agents like BaCl2.