ABSTRACT
Atualmente diversas pesquisas em Periodontia e Implantodontia visam estudar novos procedimentos e materiais que otimizem o processo cicatricial. O reparo envolve a proliferação de várias células que atuam sob a coordenação de proteínas chamadas fatores de crescimento e/ou citocinas, nos quais muitos estudos têm se concentrado e confirmado seu papel especial no processo de reparação. Artin M é uma lectina isolada de sementes de Artocarpus integrifólia e foi utilizado no tratamento tópico de lesões por queimadura de pele proporcionando aceleração da cicatrização, e redução da necrose. Em estudos recentes, foi demonstrado que o Artin M também estimulou a proliferação de fibroblastos e a reparação tecidual de lesões em mucosa palatina de ratos. Buscando um melhor entendimento da forma de atuação desta lectina, o objetivo deste estudo foi avaliar os efeitos do Artin M na expressão gênica e produção proteica de citocinas e fatores de crescimento envolvidos no processo de reparo tecidual. Culturas primárias de fibroblastos gengivais e macrófagos de rato foram tratadas com Artin M nas concentrações de 1 ; 2,5 e 5,0 µg/ml por 4, 8, 12 e 24 h para análise da expressão gênica através da reação em cadeia da polimerase após transcrição reversa de maneira quantitativa, em tempo real (RT-qPCR) e, nas mesmas concentrações por 48 e 72h após estímulo para análise quantitativa da concentração proteica dos fatores de crescimento (VEGF e TGFß) e das citocinas pró-inflamatórias (IL-1, IL-6 e TNFα) em sobrenadante de culturas de células por meio de kits ELISA (Enzyme linked Immunosorbent Assay). Os resultados demonstraram um estímulo significativo (p<0,05; ANOVA) na expressão gênica das citocinas IL-1 e TNFα tanto pelos macrófagos como os fibroblastos. A secreção proteica dos fibroblastos gengivais demonstrou aumento nos níveis de TGFß, enquanto nos macrófagos houve aumento para TNFα. Os resultados sugerem que o Artin M atua aumentando a expressão gênica e proteica de citocinas relevantes no processo de reparação
Currently, several studies in Periodontics and Implantology seek for new procedures and material that optimize the healing process. The healing process involves the proliferation of various cells that act under the coordination of proteins called growth factors and/or cytokines, which have been focused by many researches that have confirmed their special role in the repair process. Artin M is a lectin isolated from Artocarpus integrifolia seed and used in the topical treatment of skin burn injuries, providing accelerated healing and necrosis reduction. Recently, some studies demonstrated that Artin M also stimulates fibroblast proliferation and wound healing in rat oral mucosa. Seeking a better understanding of the lectin action, the aim of this study was to evaluate the effects of Artin M on gene expression and protein production of cytokines and growth factors involved in tissue repair. Primary cultures of rat gingival fibroblasts and macrophages were treated with Artin M at concentrations of 1, 2.5, and 5.0 µg / ml for 4, 8, 12 and 24 h for gene expression analysis by quantitative polymerase chain reaction (RT-qPCR) and at the same concentrations for 48 and 72h for quantitative analysis of protein concentration of growth factors (VEGF and TGFß ) and inflammatory cytokines ( IL-1 , IL-6 and TNFα ) in culture supernatants by ELISA (Enzyme linked Immunosorbent Assay). The results demonstrated significant (p < 0.05, ANOVA ) expression of IL-1 and TNF-α by macrophages as well as fibroblasts. In relation to the protein levels, gingival fibroblasts produced increased levels of TGFß, while macrophages synthesized significant levels of TNF-α. The results suggest that Artin M has a role in the increasing gene and protein expression of relevant cytokines in the repair process
Subject(s)
Animals , Rats , Enzyme-Linked Immunosorbent Assay , Cytokines , Fibroblasts , Macrophages , Wound Healing , Mannose-Binding Lectins , Analysis of VarianceABSTRACT
Objective To investigate the expression of mannose-binding lectin (MBL) in lesions of patients with psoriasis vulgaris,and to explore the relationship between MBL and psoriasis pathogenesis.Methods Immunohistochemistry and Western blot were performed to detect the expression of MBL in lesional and normalappearing perilesional skin of 30 patients with progressive psoriasis vulgaris,as well as in normal skin of 30 healthy human controls.Statistical analysis was carried out by t test using SPSS13.0 software.Results Immunohistochemistry showed that MBL was expressed in lesional psoriatic skin,but weakly expressed or absent in normalappearing perilesional skin and normal control skin,with the relative expression level of MBL in lesional skin significantly higher than that in perilesional skin and normal control skin (0.636 7 ± 0.515 1 vs.0.416 3 ± 0.160 1 and 0.381 6 ± 0.310 9,t =2.24,2.32,respectively,both P ＜ 0.05).Western blot revealed a positive expression of MBL protein in all the skin specimens,and the expression intensity of MBL protein in lesional psoriatic skin was significandy increased compared with perilesional psoriatic skin and normal control skin (0.273 1 ± 0.129 4 vs.0.186 3 ± 0.193 1 and 0.149 2 ± 0.268 7,t =2.05,2.28,respectively,both P＜ 0.05).No significant difference was shown in the expression of MBL protein between perilesional psoriatic skin and normal control skin by immunohistochemistry (t =1.51,P ＞ 0.05) or Western blot (t =0.61,P ＞ 0.05).Conclusion There is a high expression of MBL protein in lesions of patients with psoriasis vulgaris,which may be somewhat associated with the pathogenesis of psoriasis.
ABSTRACT
Objective To determine the gene polymorphism and serum concentration of mannose-binding lectin (MBL) in patients with psoriasis,and to analyze the relationship between MBL and psoriasis.Methods Totally,67 patients with psoriasis vulgaris and 69 healthy human controls were enrolled in this study.Venous blood samples were obtained from all the subjects.Genomic DNA was extracted,and PCR-restriction fragment length polymorphism (PCR-RELP) analysis was conducted to determine the polymorphism at codon 54 of the MBL gene.Enzyme-linked immunosorbent assay was performed to measure the serum level of MBL.A chi-square goodness-of-fit test was carried out to evaluate Hardy-Weinberg equilibrium,t test to compare the serum concentration of MBL,and chi-square test to compare the frequency of genotypes and alleles of MBL gene codon 54.Results The patients with psoriasis showed higher frequency of GGC/GAC heterozygote but lower frequency of GGC/GGC homozygote (x2 =10.36,P ＜ 0.05),together with increased frequency of GAC allele but decreased frequency of GGC allele (x2 =8.31,P ＜ 0.05),at codon 54 of the MBL gene compared with the healthy controls.The variant allele GAC at codon 54 of the MBL gene was markedly associated with psoriasis (OR =3.383,95％ CI 1.585-7.211,P ＜ 0.05).The serum concentration of MBL was (2.193 7 ± 0.816 3) mg/L in patients with psoriasis,significantly lower than that in the healthy controls ((3.269 5±1.205 8) mg/L,t=6.11,P＜ 0.05).Conclusion MBL might be associated with the pathogenesis of psoriasis to some degree.
ABSTRACT
Objective To analyze the single nucleotide polymorphisms(SNPs) in promoter region,5' untranslated region,exon 1 and haplotypes of mannose binding-lectin(MBL) gene in Han nationality and Hui etheic children in China.Methods Sixty nine Hui ethnic children from Ningxia Huizu Auto.Reg.and 105 Han children from Zhejiang Prov.were enrolled in the present study.Whole blood samples(1.5 ml) were collected into potassium-EDTA tubes.SNPs in promoter region,5' untranslated region and exon 1 of MBL gene were determined by sequence analysis using BigDye Mix 3730 genetic analyzer,and genetic analysis was performed using SHEsis software.Results The variant allele frequencies at-221 sites in Han and Hui objects were 0.091 and 0.123,respectively,with no difference between the two groups(?2=0.684,P=0.408).No mutation was found at sites +223 and +239 in exon 1 of MBL gene in the study.The variant frequency at +230 site in Hui children(0.268) was significantly higher than that in Han objects(0.167,?2=5.223,P=0.022).The most common haplotype was YA,and the frequencies of YA haplotype in Han and Hui ethnic were 0.770 and 0.669,respectively,with significant difference between the two groups(?2=4.312,P=0.038).Conclusion The variant allele frequency at +230 sites in exon 1 in Hui ethnic children is higher than that in Han subjects.The most common haplotype is YA,and the frequency of YA haplotype is higher in Han children than that in Hui subjects.