Investigação da funcionalidade de haplótipos no gene Interleucina 4
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Data
2016-03-28
Autores
Anovazzi, Giovana [UNESP]
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
A Doença Periodontal (DP) tem caráter multifatorial, com influência de fatores como a presença de microrganismos periodontopatogênicos, reação do sistema imune, suscetibilidade genética do hospedeiro, hábito de fumar, stress e presença de doenças sistêmicas. O tecido periodontal inflamado produz várias citocinas, dentre elas a interleucina 4 (IL-4). Estudos realizados por este grupo identificaram que indivíduos carregando os polimorfismos -590(T/C), +33(T/C) e VNTR(I/D) no gene IL4 formando o haplótipo TCI/CCI são 5 vezes mais suscetíveis à DP (Odds Ratio ajustado= 5,3; 95% Intervalo Confiança = 2,2 -12,9), enquanto o haplótipo TTD/CTI conferiu proteção contra o desenvolvimento da DP, Odds Ratio ajustado= 0,18 (95% Intervalo Confiança = 0,04 - 0,88). Assim, para verificar a funcionalidade dos haplótipos no gene IL4, o objetivo deste estudo foi investigar possíveis diferenças na resposta imune frente a estímulos inflamatórios e de bactérias periodontopatogênicas em células coletadas do sangue de pacientes que carregam tais diferentes haplótipos. A funcionalidade dos referidos haplótipos no gene IL4 foi também investigada por meio da regulação da expressão do gene repórter presente em plasmídeos recombinantes construídos artificialmente (constructos). Foram coletados sangue periférico de 5 pacientes de cada haplótipo e estimulados com mediadores inflamatórios e bactérias periodontopatogênicas para se avaliar a expressão gênica (mRNA, RT-qPCR), proteínas secretadas (Multiplex) e proteínas intracelulares caracterizando o perfil fenotípico celular (Citometria de fluxo). Constructos contendo cada haplótipo foram transfectados em células JM e suafuncionalidade foi avaliada por meio da expressão do gene repórter GFP utilizando citometria de fluxo. Independente do tipo de estímulo à que as culturas celulares foram submetidas, foram detectados níveis mais elevados de mRNA e da proteína IL-4 no grupo do haplótipo CTI/TTD, que também apresentaram níveis mais elevados de outras citocinas anti-inflamatórias. Culturas celulares com o haplótipo TCI/CCI mostraram resultado oposto; isto é, níveis mais elevados de citocinas pró-inflamatórias. Observou-se também que o constructo TCI apresentou os maiores níveis de expressão de GFP em comparação ao constructo contendo apenas o polimorfismo na região promotora. Conclui-se que os haplótipos formados por polimorfismos no gene IL4 influenciaram os níveis de expressão genica e de proteínas.
Periodontal disease (PD) is multifactorial, with the influence of factors such as the presence of periodontopathogenic micro-reaction of the immune system, genetic susceptibility of the host, smoking, stress and presence of systemic diseases. The inflamed periodontal tissue produces various cytokines, among which interleukin 4 (IL- 4). Studies by this group found that subjects carrying the -590 polymorphism (T/C) +33 (T/C) and VNTR (I/D) in the IL4 gene haplotype forming the TCI/CCI are 5 times more susceptible to PD (ORadjusted= 5.3; 95% CI = 2.2 - 12.9), while the TTD/CTI haplotype conferred protection against the development of PD (ORadjusted= 0.18; 95% CI = 0.04- 0.88). So, to check the functionality of the haplotypes in the IL4 gene, the aim of this study is to investigate possible differences in the immune response against inflammatory and periodontal bacteria stimuli in collected blood cells from patients with different haplotypes. The functionality of these haplotypes in the IL4 gene will also be investigated by means of regulation of expression of the reporter gene present in the recombinant plasmids constructed artificially (constructs). Peripheral blood was collected from 5 patients of each haplotype and stimulated with inflammatory mediators and periodontal bacteria to assess the gene expression (mRNA RT-qPCR), secreted proteins (Multiplex) and intracellular proteins featuring cellular phenotypic profile (flow cytometry). Constructs containing each haplotype were transfected into cells JM and its functionality was evaluated by GFP reporter gene expression using flow cytometry. Regardless of the type of stimulation to which the cell cultures were subjected they were detected higherlevels of mRNA and protein of IL-4 haplotype in the group CTI/TTD, which also showed higher levels of other anti-inflammatory cytokines. Cell cultures to the haplotype TCI/CCI showed the opposite result; i.e., higher levels of pro-inflammatory cytokines. It was also observed that the TCI construct showed the highest levels of GFP expression compared to the construct containing only the polymorphism in the promoter region. In conclusion the haplotypes formed by polymorphisms in the IL4 gene influenced the gene expression levels and protein.
Periodontal disease (PD) is multifactorial, with the influence of factors such as the presence of periodontopathogenic micro-reaction of the immune system, genetic susceptibility of the host, smoking, stress and presence of systemic diseases. The inflamed periodontal tissue produces various cytokines, among which interleukin 4 (IL- 4). Studies by this group found that subjects carrying the -590 polymorphism (T/C) +33 (T/C) and VNTR (I/D) in the IL4 gene haplotype forming the TCI/CCI are 5 times more susceptible to PD (ORadjusted= 5.3; 95% CI = 2.2 - 12.9), while the TTD/CTI haplotype conferred protection against the development of PD (ORadjusted= 0.18; 95% CI = 0.04- 0.88). So, to check the functionality of the haplotypes in the IL4 gene, the aim of this study is to investigate possible differences in the immune response against inflammatory and periodontal bacteria stimuli in collected blood cells from patients with different haplotypes. The functionality of these haplotypes in the IL4 gene will also be investigated by means of regulation of expression of the reporter gene present in the recombinant plasmids constructed artificially (constructs). Peripheral blood was collected from 5 patients of each haplotype and stimulated with inflammatory mediators and periodontal bacteria to assess the gene expression (mRNA RT-qPCR), secreted proteins (Multiplex) and intracellular proteins featuring cellular phenotypic profile (flow cytometry). Constructs containing each haplotype were transfected into cells JM and its functionality was evaluated by GFP reporter gene expression using flow cytometry. Regardless of the type of stimulation to which the cell cultures were subjected they were detected higherlevels of mRNA and protein of IL-4 haplotype in the group CTI/TTD, which also showed higher levels of other anti-inflammatory cytokines. Cell cultures to the haplotype TCI/CCI showed the opposite result; i.e., higher levels of pro-inflammatory cytokines. It was also observed that the TCI construct showed the highest levels of GFP expression compared to the construct containing only the polymorphism in the promoter region. In conclusion the haplotypes formed by polymorphisms in the IL4 gene influenced the gene expression levels and protein.
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Palavras-chave
Doenças periodontais, Polimorfismos genéticos, Expressão gênica, Periodontal diseases, Gene polymorphism, Gene expression