RESUMO
PURPOSE: Mitotically inhibited 3T3 fibroblasts are used as feeder layers to culture a variety of cells. However, transplantation of human cells cultured on mitotically arrested mouse cells poses potential risks, such as disease transfer and contamination with 3T3 cells. Bovine RPE and IPE cells were cultured on mitomycin-treated 3T3 fibroblasts, to examine cell characteristics and contamination by 3T3 products. METHODS: IPE or RPE cells cultured on mitomycin-treated 3T3 fibroblasts were evaluated for adhesion, morphology, and tight junction formation by microscopy and immunohistochemistry. ROS phagocytosis was used to examine functional activity. Gene expression was evaluated by quantitative real-time PCR. RESULTS: In the presence of 3T3 fibroblasts, primary IPE and RPE cells adhere, spread and acquire a hexagonal shape within 12 hours. When cultured on 3T3 fibroblasts, IPE and RPE cells exhibited stable expression of pigment epithelial genes, but expression of mouse collagen type I was also observed. CONCLUSIONS: Culturing IPE and RPE cells on mitomycin-treated 3T3 fibroblasts resulted in rapid adhesion and growth of primary pigment cells. However, the presence of potentially hazardous xenogeneic mRNA of mouse origin in the cultures limits the use of these cells for transplantation.
Assuntos
Fibroblastos/citologia , Iris/citologia , Mitose/efeitos dos fármacos , Epitélio Pigmentado Ocular/citologia , RNA Mensageiro/metabolismo , Epitélio Pigmentado da Retina/citologia , Alquilantes/farmacologia , Animais , Bovinos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular , Técnicas de Cocultura , Colágeno/genética , Colágeno Tipo I , Fibroblastos/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Perfilação da Expressão Gênica , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Mitomicina/farmacologia , Células NIH 3T3 , Fagocitose/fisiologia , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Epitélio Pigmentado Ocular/fisiologia , Epitélio Pigmentado da Retina/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Segmento Externo da Célula Bastonete/metabolismo , Suínos , Junções Íntimas , Proteína da Zônula de Oclusão-1RESUMO
BACKGROUND: Sibutramine, a centrally acting noradrenaline and serotonin re-uptake inhibitor, enhances satiety and is frequently used to support weight loss. However, a significant variability exists among individuals concerning the response to sibutramine. METHODS: We genotyped 111 participants of a randomized placebo-controlled clinical trial for the GNB3 C825T polymorphism and analysed associations of genotypes with treatment outcome. Patients undergoing a structured weight loss programme were treated with either placebo or 15 mg sibutramine daily for 54 weeks. RESULTS: In the placebo group, the non-pharmacological programme alone resulted in a significantly greater weight loss in individuals with the GNB3 TT/TC genotypes as compared to individuals with the CC genotype (-7.1 +/- 1.2 vs. -2.7 +/- 1.5 kg, P = 0.031). Administration of 15 mg sibutramine was more effective in individuals with the CC genotype than in the subjects with the TT/TC genotypes (weight loss: 7.2 +/- 2.2 vs. 4.1 +/- 2.1 kg, P = 0.0013, sibutramine vs. placebo). In the CC genotype carriers, the odds ratio (OR) for a weight loss greater than 5% (sibutramine vs. placebo) was 6.6 (95% CI 1.8-25.6; P = 0.004) and for a weight loss greater than 10% was 9.6 (95% CI 1.7-53.8; P = 0.010). CONCLUSION: Genotyping for the GNB3 C825T polymorphism is highly predictive for the identification of obese individuals who will benefit from sibutramine treatment.
