The Study Of Impaired Apoptosis Function Of Fas And P53 Protein In The Fibroblasts Derived From Keloid

BackgroundAccumulating evidence indicates that unbalance between proliferation and apoptosis of fibroblasts contributes mainly to the formation of keloid and hypertrophic scar. So far, treatment for keloid is less effective. Fibroblasts from keloid are know to be abnormal ones, their apoptosis function was impaired, thus it is necessary for us to find out the cause, in which apoptotic function was impaired.Our previous study has demonstrated that P53 and Fas were involved in keloid formation. Detection of polymorphisms in p53 gene codon 72 was important for deciding who are susceptible to keloid. Monoclonal antibody against Fas at a dose of ng-μg induced the apoptosis in Proliferative fibroblasts derived from hypertrophic scar, not in the fibroblasts from keloid. Fas protein expression was decreased in hypertrophic scar and increased in keloid. Abnormal apoptosis occurred in keloid was not associated with the loss of Fas receptors. Fas-mediated death signals were blocked at the upstream. Transfection of normal fas gene into target cells could improve the unbalance of proliferation and apoptosis in the fibroblast.Polymorphisms in p53 gene codon 72 were intensively investigated. Different gene types of p53 are involved in the development of tumor. The products expressed by different gene typed of P53 may affect the cell apoptosis of fibroblast. However, Polymorphisms in p53 gene and protein expression in the local tissue from keloid are unclear. ObjectiveThe present study was done to observe the relationship between clinical phenotype of keloid and the polymorphisms in p53 gene codon 72, and explode the role of gene variation of p53 in the keloid formation and possible mechanism of impaired apoptosis. Meanwhile, we also investigated the effects of cisplatin and hypoxia on the apoptosis of fibroblasts from keloid.Methods1. The tissue and blood samples were taken from patients with keloid (n=35) and healthy individuls (n=26). The diagnosis of keloid was based on clinical manifestation, histological examination. The samples from patients with keloids were divided into three groups according to clinical phenotype. DNA of the tissue and blood samples was extracted. The tissue samples of the keloid were divided into peripheral and central areas. The expression of P53 in protein levels was detected by immunostaining, and DNA sequencing was performed with sequence-based typing (SBT).2. Fibroblasts from hypertrophic scar and keloid were cultured. Western Blot and Flow cytometry were used to detect the effect of turicamycin on Fas protein expression and FasMcAb-induce apoptosis.3. Cisplatin and hypoxia induced the apoptosis of fibroblasts from keloid. Flow cytometry were used to detect the rate of apoptois. Western Blot was used to examine the expression of Bcl-2 and Bax.Results1. Immunostaining results revealed that P53 was detectable in all the keloid samples. Three types of gene variation of p53 codon 72, including Pro/Pro, Pro/Arg, Arg/Arg were observed in keloids, and associated with clinical phenotype of keloids. Arg polymorphic variants of P53 were increased from the peripheral area to center of keloids. The gene type of p53 codon 72 in normal skin were Arg/Arg in eighteen samples, Pro/Arg in six samples. P53 expression in keloids was mainly located in the cytoplasm of the fibroblasts, and showed positive staining in all samples detected in the study. P53 expression was not detectable in normal skin.2. The glycosylation level in Fas protein was higher in hypertrophic scar than in keloid. FasMcAb induced the apoptosis in cultured fibroblast, this action positively correlated with the glycosylation level in Fas protein. Treatment with turicamycin in cultured fibroblast decreased markedly the glycosylation level of Fas protein, and FasMcAb-induced apoptosis.3. Hypoxia did not affect the apoptosis and Bax protein expression in cultured fibroblasts derived from keloid. Treatment with cisplatin in cultured fibroblast from keloid increased the apoptosis, downregulated Bcl-2 expression and upregulated Bax expression.Conclusion:1. There are possible associations between the codon 72 polymorphism and the clinical phenotype of keloids, the alteration of codon 72 polymorphism might be beneficial to apoptosis of fibroblasts in keloids.2. FasMcAb-induced apoptosis in the cultured fibroblast correlated positively with the glycosylation level in Fas protein. Turicamycin decreased markedly the glycosylation level in Fas protein.3. Cisplatin increased the apoptosis in cultured fibroblast, which might be associated with the altered expression in Bcl-2 and Bax.