The Expression Of Nuclear Factor-kappaB And Zinc Finger Protein A20 In Keloid

Keloid represent a dysregulated response to cutaneous wound healing, and extend beyond the borders of the original wound. They are characterized by high fibroblasts activities and excessive deposition of extracellular matrix. The exact pathophysiologic mechanism for keloid formation remains largely unknown, so there is no one modality that is always successful. Keloid has so far been a headache to the patients and a challenge to orthopedists. Studies show fibroblasts are the initial cells that are involved in the changes in keloid fibroblasts'gene expression, proliferation apoptosis, synthesis and secretion as well as collage metabolism, which in turn causes fibroblasts function differently from normal skin fibroblasts.Nuclear factor-κB (NF-κB) is an ubiquitous transcription factor in mammalian cells, and critically regulate the expression of different genes in cells with its signal transduction pathway get involved in the whole biological process of wound healing. It funtions as a transmitter when signals transfer into cells, playing a role in their immune reaction, inflammatory reaction, cell proliferation, apoptosis and other cell stress reactions. NF-kB is a kind of protein dimer composed of rel family protein, commonly being p50/p65 hetero dimers. It mainly mediates the combination of rel protein and DNA. When cells are resting, p65 subunit of NF-kB is restricted to cytoplasm by inhibitory protein LkBs and fail to transcribe and regulate; If stimulated by external signals, inhibitory protein LkB is dissociated from NF-kB and enters into cell nuclear combining with a particular DNA to transcribe and regulate.A20 gene, short for Zin-Finger Protein A20 (ZFPA20) as a kind of negative control protein, has been found in the research of cellular endogenous protective mechanism. It is confirmed that Zinc Finger Protein A20 is the important regulation protein for inhibiting the activation of NF-κB. But so far, there has been very little study about NF-kB signal transduction pathway in keloids and no research about expressions changes of ZFPA20 and their relationship with keloids.In this study by using immunohistochemistry and real time quantitative RT-PCR method, we detected the protein expression and the mRNA expression of NF-kB and Zinc Finger Protein A20 in the keloid and normal skin to investigate the relationship between these factors and the formation of keloid. It would provide new aims for preventing the formation of keloid and exploring the target in treating keloid.Objective1 To investigate the protein expression of NF-κB and Zinc Finger Protein A20 in the keloid and normal skin and explore its underlying molecular biological pathogeny.2 To explore the growth and proliferation of keloid fibroblasts and normal skin fibroblasts when they were cultured in vitro and to determine their time of entering logarithmic growth phase.3 To investigate the mRNA expression of NF-κB and Zinc Finger Protein A20 in the fibroblasts derived from keloid and normal skin when both cultured in vitro.4 To observe the growth and morphology of the fibroblasts derived from normal skin and keloid.5 To study the activation of NF-kB and its relationship to formation of keloids, which could help provide new ideas about prevention and treatment of keloid..MaterialThe tissues of keloid were collected from auricular lobule, chest and perineum of 16 patients, of whom were 6 males and 10 femals. The age of patients ranged from 18 to 42 years old (average age:32.60 years old). Course of treatment:6 months to 10 years(mean:3.40years), keloid extend beyond'the original area of skin injury. Sixteen cases of normal skin collected from abdomen and limbs were used as control. There were 9 males and 7 females. The age of patients ranged from 2 to 54 years old (mean: 23.54 years old).Methods1 Tissues of normal skin and keloids were collected to make paraffin sections and explored the difference between the expressions of A20 and NF-kB in both through immunocytochemical straining.2 By tissue particle adhering, the fibroblasts from keloid and normal skin were cultured in vitro. Their growth curves were drawn and assayed by cell counting method so as to evaluate the growth proliferation of cells and observe their time of entering logarithmic growth phase.3 Take keloid fibroblasts and normal skin fibroblasts as research subjects, and put the 4th generation of the fibroblasts under experiment. By using RT-PCR(reverse transcription-polymerase chain reaction) to detect the expressions of NF-kB and ZFPA20 mRNA and explicit their respective expression characteristics in the fibroblasts cultured in vitro.Results1 Expression of NF-Kb p65 in both keloid tissues and normal skin tissuesImmunohistochemical straining showed NF-Kb p65 was localized in cytoplasm and nuclei, in the form of brown granules under normal condition and almost nuclear straining; after activation it entered into the nuclear and strained.The protein expression of NF-κB p65 were localized in the nuclei. The positive rate in keloid tissues and normal skin were tissues 87.50%(14/16) and 6.25%(1/16). There were significant difference between the two groups (p<0.05).2 The protein expression of Zinc Finger Protein A20Immunohistochemical straining'showed ZFPA20 was localized in the cytoplasm, in the form of brown granules. The positive rate of ZFPA20 in the cytoplasm of keloid fibroblasts was 18.75%(3/16); the positive rate of ZFPA20 in the cytoplasm of normal skin fibroblasts was 87.50%(14/16). There were significant difference between the two groups (p<0.05).3 Growth curves comparison of keloid fibroblasts and normal skin fibroblasts cultured in vitroThere was no remarkable difference between the growth of the two kinds of fibroblasts and the time of their entering into logarithmic growth phase was approximately same.4 Cryopreservation resuscitation of fibroblast cultured in vitroAfter the fibroblasts cultured in vitro were cryopreserved in liquid nitrogen, cells resuscitation was good. Most cells post inoculation grow well with a survival rate 85% and no remarkable changes of cell morphology was observed through inverted phase contrast microscope.5 NF-kB mRNA expression in keloid fibroblasts and normal skin fibroblastsReal-time RT-PCR assay indicated NF-kB mRNA expression level in normal skin fibroblasts was remarkably higher than that in keloid fibroblasts and there was significant difference between them (p<0.05).6 Zinc Finger Protein A20 (ZFPA20) mRNA expression in keloid fibroblasts and in normal skin fibroblastsReal-time RT-PCR assay showed the mRNA expression transcription level of Zinc Finger Protein A20 in keloid tissues were lower than those in normal skin tissues(p<0.05).Conclusion1 These related factors involved in NF-κB signal pathways showed abnormal expression in keloid. NF-κB signal pathways could pfay a role in keloid pathogenesis.2 The mRNA expression of Zinc Finger Protein A20 in keloid tissues was lower than that in normal skin tissues, which can promote the formation of keloid and indicated its low level expression could be related to its reducing inhibition of NF-kB.3 The fibroblasts isolated from norml skin and keloid exhibited similar morphology and growth rates. And the time of their entering into logarithmic phase was approximately same.4 The fibroblasts from norml skin and keloid were frozen and resuscitated successfully.