RESUMEN

MAIN CONCLUSION: We generated transplastomic tobacco lines that stably express a human Basic Fibroblast Growth Factor (hFGFb) in their chloroplasts stroma and purified a biologically active recombinant hFGFb. MAIN: The use of plants as biofactories presents as an attractive technology with the potential to efficiently produce high-value human recombinant proteins in a cost-effective manner. Plastid genome transformation stands out for its possibility to accumulate recombinant proteins at elevated levels. Of particular interest are recombinant growth factors, given their applications in animal cell culture and regenerative medicine. In this study, we produced recombinant human Fibroblast Growth Factor (rhFGFb), a crucial protein required for animal cell culture, in tobacco chloroplasts. We successfully generated two independent transplastomic lines that are homoplasmic and accumulate rhFGFb in their leaves. Furthermore, the produced rhFGFb demonstrated its biological activity by inducing proliferation in HEK293T cell lines. These results collectively underscore plastid genome transformation as a promising plant-based bioreactor for rhFGFb production.

Asunto(s)

RESUMEN

ABSTRACT Purpose: To assess the effects of the preoperative application of artificial tears combined with recombinant bovine basic fibroblast growth factor on the ocular surface function and inflammatory factor levels after operation in cataract patients complicated with dry eyes. Methods: A total of 118 cataract patients (118 eyes) complicated with dry eyes treated from February 2019 to February2020 were assigned to control and observation groups (n=59 eyes/group) using a random number table. One week before the operation, the control group was administered 0.1% sodium hyaluronate eye drops (artificial tears), based on which the observation group received Beifushu eye drops (recombinant bovine basic fibroblast growth factor), both 6 times daily for 1 week. A comparison was made between the scores of clinical symptoms and the indices of ocular surface function, inflammatory factors in tears, and oxidative stress indices before and after the operation. The ocular surface function was evaluated by an ocular surface disease index questionnaire, tear film breakup-time assay, Schirmer's I test, and corneal fluorescein stain test. The inflammatory factors in tears were measured. Results: No significant differences were noted in the general data and clinical symptom score, ocular surface disease index, tear film breakup-time, Schirmer's I test score, fluorescein stain score, interleukin-6, tumor necrosis factor-alpha, malondialdehyde, superoxide dismutase, lipid peroxide, and total antioxidant capacity before treatment between the 2 groups (p>0.05). After treatment, the clinical symptom score, ocular surface disease index, fluorescein stain score, tumor necrosis factor-alpha, interleukin-6, malondial-dehyde and lipid peroxide declined significantly, and tear film breakup-time, Schirmer's I test score, superoxide dismutase, and total antioxidant capacity increased in both the groups. The improvements in the clinical symptom score as well as in the indices of ocular surface function, inflammatory factors, and oxidative stress were more prominent in the observation group than in the control group (p<0.05). Conclusions: Artificial tears combined with recombinant bovine basic fibroblast growth factor before operation. significantly improved the ocular surface function, reduced inflammatory factors in tears, and alleviated dry eye symptoms after operation in cataract patients.

RESUMO Objetivo: Avaliar os efeitos da aplicação pré-operatória de lágrimas artificiais combinadas com o fator de crescimento de fibroblastos básicos bovinos recombinantes na função da superfície ocular e níveis de fator inflamatório após cirurgia em pacientes com catarata complicada com olhos secos. Métodos: Um total de 118 pacientes com catarata complicada com olhos secos (118 olhos), tratados entre fevereiro de 2019 e fevereiro de 2020, foram divididos em grupos de controle e de observação (n=59, 59 olhos) usando uma tabela de números aleatórios. Uma semana antes da cirurgia, o grupo controle recebeu colírio de hialuronato de sódio a 0,1% (lágrimas artificiais), enquanto o grupo de observação recebeu colírio Beifushu (fator de crescimento de fibroblastos básicos bovinos recombinantes), ambos, seis vezes ao dia, por uma semana. Antes do tratamento e um mês após a cirurgia, os escores de sintomas clínicos, índices de função da superfície ocular, níveis de fatores inflamatórios nas lágrimas e índices de estresse oxidativo foram comparados. A função da superfície ocular foi avaliada pelo questionário do índice de doença da superfície ocular, ensaio de tempo de ruptura do filme lacrimal, teste I de Schirmer e teste de coloração por fluoresceína da córnea. Os níveis de fatores inflamatórios nas lágrimas foram medidos. Resultados: Não houve diferenças significativas nos dados gerais e no escore de sintomas clínicos, índice de doença da superfície ocular, tempo de ruptura do filme lacrimal, escore do teste I de Schirmer, pontuação do teste de coloração por fluoresceína da córnea, interleucina-6, fator de necrose tumoral alfa, malondialdeído, superóxido dismutase, peróxido lipídico e capacidade antioxidante total antes do tratamento entre os dois grupos (p>0,05). Após o tratamento, o escore de sintomas clínicos, índice de doença da superfície ocular, escore do teste de coloração por fluoresceína da córnea, fator de necrose tumoral alfa, interleucina-6, malondialdeído e peróxido lipídico diminuíram significativamente, e o tempo de ruptura do filme lacrimal, escore do teste I de Schirmer, superóxido dismutase e a capacidade antioxidante total aumentou em ambos os grupos. As melhorias no escore de sintomas clínicos, bem como os índices de função da superfície ocular, fatores inflamatórios e estresse oxidativo foram mais proeminentes no grupo de observação do que no grupo controle (p<0,05). Conclusões: Lágrimas artificiais combinadas com fator de crescimento de fibroblastos básicos recombinantes antes da cirurgia melhoram notavelmente a função da superfície ocular, diminuem os níveis de fatores inflamatórios nas lágrimas e aliviam os sintomas de olho seco após a cirurgia em pacientes com catarata complicada com olhos secos.

RESUMEN

Abstract Introduction Postoperative dysphonia is mostly caused by vocal fold scarring, and careful management of vocal fold surgery has been reported to reduce the risk of scar formation. However, depending on the vocal fold injury, treatment of postoperative dysphonia can be challenging. Objective The goal of the current study was to develop a novel prophylactic regenerative approach for the treatment of injured vocal folds after surgery, using biodegradable gelatin hydrogel microspheres as a drug delivery system for basic fibroblast growth factor. Methods Videoendoscopic laryngeal surgery was performed to create vocal fold injury in 14 rabbits. Immediately following this procedure, biodegradable gelatin hydrogel microspheres with basic fibroblast growth factor were injected in the vocal fold. Two weeks after injection, larynges were excised for evaluation of vocal fold histology and mucosal movement. Results The presence of poor vibratory function was confirmed in the injured vocal folds. Histology and digital image analysis demonstrated that the injured vocal folds injected with gelatin hydrogel microspheres with basic fibroblast growth factor showed less scar formation, compared to the injured vocal folds injected with gelatin hydrogel microspheres only, or those without any injection. Conclusion A prophylactic injection of basic fibroblast growth factor -containing biodegradable gelatin hydrogel microspheres demonstrates a regenerative potential for injured vocal folds in a rabbit model.

Resumo Introdução A disfonia pós-operatória é causada principalmente por cicatrizes nas pregas vocais. Tem sido relatado que o manejo cuidadoso da cirurgia das pregas vocais reduz o risco de formação de cicatriz. No entanto, a depender da lesão da prega vocal, o tratamento da disfonia pós-operatória pode ser desafiador. Objetivo Desenvolver uma nova abordagem regenerativa profilática para o tratamento de pregas vocais lesionadas após a cirurgia, com microesferas biodegradáveis de hidrogel de gelatina como sistema de administração de medicamentos para o Fator Básico de Crescimento de Fibroblastos (bFGF). Método A cirurgia laríngea videoendoscópica foi feita para criar lesão nas pregas vocais em 14 coelhos. Imediatamente após esse procedimento, microesferas biodegradáveis de hidrogel de gelatina com bFGF foram injetadas na prega vocal. Duas semanas após a injeção, as laringes foram excisadas para avaliação da histologia das pregas vocais e do movimento da mucosa. Resultados A presença de função vibratória deficiente foi confirmada nas pregas vocais lesionadas. A histologia e a análise de imagem digital demonstraram que as pregas vocais lesionadas injetadas com microesferas de hidrogel de gelatina com bFGF apresentaram menor formação de cicatriz, em comparação com as pregas vocais lesionadas injetadas apenas com microesferas de hidrogel de gelatina ou aquelas sem injeção. Conclusão Uma injeção profilática de microesferas biodegradáveis de hidrogel de gelatina com bFGF demonstra um potencial regenerativo para pregas vocais lesionadas em um modelo de coelho.

Asunto(s)

RESUMEN

INTRODUCTION: Postoperative dysphonia is mostly caused by vocal fold scarring, and careful management of vocal fold surgery has been reported to reduce the risk of scar formation. However, depending on the vocal fold injury, treatment of postoperative dysphonia can be challenging. OBJECTIVE: The goal of the current study was to develop a novel prophylactic regenerative approach for the treatment of injured vocal folds after surgery, using biodegradable gelatin hydrogel microspheres as a drug delivery system for basic fibroblast growth factor. METHODS: Videoendoscopic laryngeal surgery was performed to create vocal fold injury in 14 rabbits. Immediately following this procedure, biodegradable gelatin hydrogel microspheres with basic fibroblast growth factor were injected in the vocal fold. Two weeks after injection, larynges were excised for evaluation of vocal fold histology and mucosal movement. RESULTS: The presence of poor vibratory function was confirmed in the injured vocal folds. Histology and digital image analysis demonstrated that the injured vocal folds injected with gelatin hydrogel microspheres with basic fibroblast growth factor showed less scar formation, compared to the injured vocal folds injected with gelatin hydrogel microspheres only, or those without any injection. CONCLUSION: A prophylactic injection of basic fibroblast growth factor -containing biodegradable gelatin hydrogel microspheres demonstrates a regenerative potential for injured vocal folds in a rabbit model.

Asunto(s)

RESUMEN

The effects of deslorelin acetate use in inducing ovulation need to be clarified to improve the results of equine embryo transfer. The mRNA abundance for angiogenic factors and LH receptor (LHR) in corpus luteum (CL) was studied in mares with natural (control group [CG]) and induced ovulation with deslorelin acetate (treatment group [TG]; follicles: ≥ 35 mm). Transrectal ultrasonography was used to verify the ovulation day, and on Days 4, 8, and 12 after ovulation (Day 0), CL samples were obtained through ultrasound-guided biopsy. The messenger RNA expression of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and LHR genes were analyzed by real-time polymerase chain reaction. A positive correlation was observed between VEGF and LHR (P < 0.00001, r = 0.78), and it was possible to detect higher LHR expression in the TG than in the CG on Day 4 (P < 0.05). Moreover, this expression was higher on Days 4 and 8 than on Day 12 in the TG. Basic fibroblast growth factor was also expressed in luteal tissue on all days for both groups; however, these differences were not significant. In conclusion, deslorelin acetate was effective for the induction of ovulation in mares, resulting in higher expression of LHR, especially on the fourth day after ovulation. In addition, VEGF expression was influenced by induced ovulation, with a lower level on Day 12, which is expected in nonpregnant mares.

Asunto(s)

RESUMEN

Administration or expression of growth factors, as well as implantation of autologous bone marrow cells, promote in vivo angiogenesis. This study investigated the angiogenic potential of combining both approaches through the allogenic transplantation of bone marrow-derived mesenchymal stem cells (MSCs) expressing human basic fibroblast growth factor (hbFGF). After establishing a hind limb ischemia model in Sprague Dawley rats, the animals were randomly divided into four treatment groups: MSCs expressing green fluorescent protein (GFP-MSC), MSCs expressing hbFGF (hbFGF-MSC), MSC controls, and phosphate-buffered saline (PBS) controls. After 2 weeks, MSC survival and differentiation, hbFGF and vascular endothelial growth factor (VEGF) expression, and microvessel density of ischemic muscles were determined. Stable hbFGF expression was observed in the hbFGF-MSC group after 2 weeks. More hbFGF-MSCs than GFP-MSCs survived and differentiated into vascular endothelial cells (P<0.001); however, their differentiation rates were similar. Moreover, allogenic transplantation of hbFGF-MSCs increased VEGF expression (P=0.008) and microvessel density (P<0.001). Transplantation of hbFGF-expressing MSCs promoted angiogenesis in an in vivo hind limb ischemia model by increasing the survival of transplanted cells that subsequently differentiated into vascular endothelial cells. This study showed the therapeutic potential of combining cell-based therapy with gene therapy to treat ischemic disease.

Asunto(s)

RESUMEN

Antecedentes: El trasplante traqueal de longitudes mayores a 6 cm de longitud ha fallado por complicaciones isquémicas del injerto. Experimenta/mente se han utilizado factores de crecimiento y diferentes técnicas quirúrgicas, como la de trasplante traqueal dividido para favorecer la neoformación de vasos sanguíneos. Objetivo: Evaluar la viabilidad, cambios tráquea cervical, macroscópicos y microscópicos del trasplante de tráquea cervical en perros al utilizar la técnica quirúrgica de trasplante traqueal dividido, combinando la aplicación del factor básico de crecimiento para fibroblastos en los sitios de las anastomosis. Material y métodos: Se operaron 24 perros que fueron divididos en 4 grupos de estudio: Grupo I (n = 6): Trasplante de 9 anillos de tráquea cervical con la técnica convencional (TTCC); Grupo II (n = 6): TTCC e instilación tópica de factor básico de crecimiento de fibroblastos (bFGF) en los sitios de anastomosis; Grupo III (n = 6): Trasplante con la técnica de trasplante dividido (TTCD) y Grupo IV (n = 6): TTCD y aplicación de bFGF. Los animales recibieron triple inmunosupresión (azatioprina, metilprednisolona, ciclos-porína). Se planearon evaluaciones clínica, radiológica y traqueoscópica durante 4 semanas. Al final del estudio los injertos trasplantados se evaluaron macroscópica y microscópicamente. Resultados: Ningún animal concluyó su tiempo de estudio por disnea grave durante la primera semana del estudio. Macroscópicamente todos los injertos desarrollaron fístulas y necrosis. Microscópicamente mostraron necrosis, inflamación, vasculitis, hemorragia y destrucción del cartílago. Conclusión: En este estudio encontramos que el trasplante de tráquea cervical mayor a 6 cm, tiene malos resultados con la técnica quirúrgica convencional o dividida e inmunosupresión, así como con y sin la aplicación de bFGF en los sitios de anastomosis.

Background: Tracheal transplantation of lesions larger than 6 cm fails due to ischemic complications. Growth factors and different surgical techniques, including the divided tracheal graft technique, have been used experimentally to stimulate the neoformation of blood vessels. Objective: Assessment of the viability and macroscopic and microscopic changes of the cervical transplanted trachea in dogs, using the divided tracheal graft technique, combined with the application of basic fibroblast growth factor (bFGF) on the anastomotic site. Material and methods: Twenty four mongrel dogs were divided in 4 study groups: Group I (n = 6): Transplantation of 9 cervical tracheal rings with the conventional surgical technique (TCTC). Group II (n = 6): TCTC combined with topical instillation of bFGF at on the anastomotic line. Group III (n = 6): Transplantation of cervical trachea using the divided tracheal graft technique (TCTD), and Group IV (n = 6): TCTD with topical application of bFGF. The animals received triple immunotherapy (azathioprine, methylprednisolone, cyclosporine) and were to have clinical, radiological and endoscopical evaluation during 4 weeks. At the end of the study, macroscopic and microscopic evaluations of the transplanted grafts were done. Results: No animal completed the study time due to severe dyspnea during the first postoperative week. Macroscopically all grafts showed necrosis and fistulae formation. Microscopically we observed necrosis, inflammation, vasculitis, hemorrhage and destruction of cartilage in all the grafts. Conclusion: In this study, tracheal allotransplanta-tion longer than 6 cm with either surgical technique, with immunosuppression, with or without bFGF, is unsuccessful.

RESUMEN

The growth of cells is controlled by stimulatory or inhibitory factors. More than twenty different families of polypeptide growth factors have been structurally and functionally characterized. Basic fibroblast growth factor (bFGF) of the fibroblast growth factor family was characterized in 1974 as having proliferative activity for fibroblastic cells. The inhibitory effects of ethanol on cell proliferation result from interference with mitogenic growth factors (e.g., bFGF, EGF and PDGF). In order to better understand the mode of action of bFGF, particularly regarding the influence of ethanol on the biological activity of bFGF, three recombinant bFGF mutants were produced (M6B-bFGF, M1-bFGF and M1Q-bFGF). In the present study, wild bFGF and these mutants were examined by molecular dynamics simulations in systems consisting of a solute molecule in ethanol solution at 298 K and physiological pH over 4.0 ns. The hydrogen bonds, the root mean square deviations and specific radial distribution functions were employed to identify changes in the hydrogen bond structures, in the stability and in the approximation of groups in the different peptides to get some insight into the biological role of specific bFGF regions. The detailed description of the intramolecular hydrogen bonds, hydration, and intermolecular hydrogen bonds taking place in bFGF and its mutants in the presence of ethanol established that the residues belonging to the beta5 and beta9 strands, especially SER-73(beta5), TYR-112(beta9), THR-114(beta9), TYR-115(beta9), and SER-117(beta9), are the regions most affected by the presence of ethanol molecules in solution.

Asunto(s)

RESUMEN

O objetivo do presente estudo avaliar o efeito da combinação do fator de crescimento de fibroblastos básico (b-FGF) e fator de transformação de crescimento beta (TGFbeta) na proliferação, expressão de genes para colágeno tipos I e III, metaloproteases -1 e ­2 e TIMPs 1, 2 e 3, e na síntese destes próprios fatores de crescimento pelas células do ligamento periodontal de humanos. Para a realização do estudo, as células em diferentes concentrações, de acordo com cada experimento, foram tratadas com os fatores de crescimento nas concentrações de 1.0ng e 10ng/ml de meio de cultura para o b-FGF e para o TGF-ß, tanto isoladamente quanto associados. A avaliação da proliferação celular foi realizada após os períodos de 24 e 48h na presença dos fatores de crescimento, através da mensuração do nível de MTS reduzido a formazan pelas células viáveis, e a síntese de b-FGF e TGF-ß pelo teste ELISA empregando a técnica "sandwich". A expressão de genes para colágeno tipos I e III, metaloproteases-1 e -2 foi avaliada pela técnica de RT-PCR. O teste ANOVA mostrou haver diferenças estatisticamente significantes para proliferação e síntese de bFGF (p<0.05) entre os tratamentos. O teste de Comparação Múltipla de Bonferroni mostrou que todas as associações aumentaram a proliferação celular e a síntese de bFGF (p<0.001). Isoladmente, só o TGF-ß foi capaz de estimular a proliferação, independente do tempo e da dose (p<0.001), enquanto que, somente o bFGF estimulou a síntese dele próprio de maneira dose-dependente (p<0.05). Em relação à síntese de TGF-ß, apenas a associação das menores doses de cada fator, foi capaz de modular a síntese dele mesmo. O teste de Kolmogorov- Smirnov mostrou haver diferenças estatisticamente significantes quanto à expressão de genes entre o uso isolado dos fatores de crescimento (p<0.1), já que o bFGF aumentou a expressão de MMP-1 e MMP-2 e reduziu para Col I e III, e TIMPs1, 2 e 3, enquanto que, o TGF-ß atuou de maneira inversa. Todas as associações apresentaram efeito semelhante ao uso isolado do TGFß, com exceção da associação de 10ng de bFGF com 1ng de TGF-ß, na qual prevaleceu o efeito do bFGF. Conclui-se que as associações do bFGF e do TGF-ß atuaram de maneira dose-dependente no estímulo a proliferação celular, o aumento na expressão de genes para colágeno e TIMPs, e redução para as metalproteases e, modulação da síntese deles próprios

The aim of this study was to evaluate the effect of association of basic fibroblast growth factor and transforming growth factor beta on the proliferation, expression of colagen type I e III, matrix metalloproteinases-1 e ­2 e TIMPs 1, 2 e 3, and on the modulation of the syntheses oh these growth factors by humans periodontal ligament cells. Different concentrations of cells, according to experiment, were incubated on the presence of 1.0 or 10 ng/ml of DMEM of growth factors, associated or no. The cellular proliferation was evaluated to 24 and 48 hours of incubation by the colorimetric method. The synthese of bFGF and TGF-ß was verificated by ELISA method, using a "sandwich" techinique, and mRNA expression by Real Time - PCR. ANOVA test results indicated significant differences on the proliferation and bFGF synthesis (p<0.05) among the treatments. Bonferroni for pairwise comparition test results showed that all association estimulated the proliferation and bFGF synthesis (p<0.001). Alone, only TGF-ß can stimulate the proliferation to both concentrations and periods (p<0.001), and only bFGF stimulated its own synthesis by dosedependent manner (p<0.05). Only the asssociation of 1ng of each growth factor can stimulate the synthesis of TGF-ß. Kolmogorov-Smirnov test results indicated significant differences on the expression of mRNA among treatment with the isolated growth factors (p<0.1). Basic fibroblast growth factor increased the expression of MMP-1 e MMP-2 and reduced to collagen I e III, e TIMPs1, 2 e 3, as long as TGF-ß behaved the inverse manner. All the associations had similar effect to isolated TGF-ß, except the association of 10ng of bFGF to 1ng of TGF-ß. In conlusion, the associations of bFGF e TGF-ß influenced of dose-dependent manner on the cellular proliferation, on the increasing of the expression to collagen and TIMPs, and on the reduction to metaloproteinases and, the modulation of these own synthesis

Asunto(s)

RESUMEN

Endooligopeptidase A is a putative neuropeptide-metabolizing enzyme. It converts small enkephalin-containing peptides into the corresponding enkephalins and inactivates biopeptides such as bradykinin and neurotensin in vitro. We investigated the presence of endooligopeptidase A in PC12 cells. This cell line was derived from a rat pheochromocytoma tumor and resembles fetal chromaffin cell. Depending on the supplements added to the cell culture, this cell line can be differentiated into mature chromaffin cell or sympathetic neuron-like cell. Endooligopeptidase A activity was measured in soluble cellular extracts using a specific fluorogenic substrate QF-ERP7. The PC12 endooligopeptidase A-like activity shared similar but not identical biochemical properties with rabbit brain endooligopeptidase A. Similarly to rabbit brain endooligopeptidase A, the PC12 endooligopeptidase A-like activity was enhanced by DTT, totally inhibited by DTNB and 1-10 Phenanthroline, partially inhibited by cFP-AAF-pAb, and not affected by PMSF. Furthermore, the PC12 endooligopeptidase A-like activity displayed identical elution profile as rabbit brain endooligopeptidase A in gel filtration and anion-exchange chromatography. In addition, an antiserum raised against rabbit brain endooligopeptidase A cross-reacted with a 71 kDa component from PC12 cell extracts in Western blotting and was also able to partially neutralize the PC12 endooligopeptidase A-like activity. Treatment of PC12 cells with basic fibroblast growth factor (bFGF), a neurotrophic factor for this cell line, did not modify the specific activity of this enzyme. However, cAMP analogs decreased the specific activity of the enzyme. These results indicate the presence of an endooligopeptidase A-like activity in PC12 cells which is modulated by cAMP but not by bFGF.

Asunto(s)