Experimental Study Of P53 Gene Mutations In Keloids

Background: Keloids are the result of a dysregulated wound healing process. They are characterized by the formation of excess scar tissue that proliferates beyond the boundaries of the original wound. Keloids are locally aggressive, and in contrast to hypertrophic scars, they invade healthy tissue. They are much more likely to recur after treatment for removal or reduction and can actively persist for many years. In this manner, keloids behave clinically as benign tumors that form as a result of an inherited defect in wound healing. The distortion, contractures and cosmetic problems associated with keloids present a formidable clinical challenge. Keloid fibroblasts exhibit an elevated rate of collagen synthesis compared with normal mature scars and demonstrate both the reduced requirements of growth factors in vitro and the hyporesponsiveness to mediators that are inhibitory to normal fibroblasts. Despite various theories such as abnormal molecular factors or cytokines about the abnormal growth characteristics of keloid, the mechanisms of keloidogenesis remain unknown.The p53 gene on chromosome 17pl3.1 encodes a 53kd phosphoprotein that acts as a transcription factor and has tumor-suppressor functions. Evidences suggest that p53 controls a cell cycle checkpoint responsible for maintaining the integrity of the genome. It has been shown that the wild-type p53 induces cell cycle arrest and/or apoptosis in response to DMA damage. Mutations in p53 eliminate this response and result in an enhanced frequency of genomicrearrangements. It is thought that the resulting genetic instability increase the probability that the tumor cells escape the normal restrictions against excessive growth. Evidences show that a wide variety of sporadic human tumors associated with different genetic alterations are characterized by the aberrant expression of p53. Mutant p53 alleles appear to increase the risk of tumor. The relationship between p53 and the pathway of apoptosis has been well characterized, and the rate of apoptosis is drastically reduced in keloid fibroblasts compared with that in normal fibroblasts. The question may therefore be raised as to whether the p53 mutations play an important role in keloid formation.Objective: To detect p53 gene mutations in a series of human keloid tissues and cultured keloid fibroblasts. Further investigate the effect of the wild-type p53 gene on both apoptosis and cell cycle progression of keloidfibroblasts, thus characterize the role of p53 mutations in keloid formation.Methods:Part 1:12 keloid tissues and 12 normal scar tissues were obtained from 24 patents with keloid or normal scar lesions. Healthy skins and blood samples were obtained from these patients. Fibroblasts were derived and cultured from these tissues. Use Polymerase Chain Reaction-Based Single-Stand Conformational Polymorphism (PCR-SSCP) and DMA sequencing to detect the exons 4-8 of p53 gene mutations.Part 2: Fibroblasts of keloid were transfected with recombinant adenovirus containing human wide-type p53 (Ad-p53) in vitro. The expression of wide-type p53 was analysised with Northern Blot and western Blot.Part 3: Fibroblasts of keloid were transfected with recombinant adenovirus containing human wide-type p53 (Ad-p53) in vitro. The growth and apoptosis of transfected cells were studied by cell count and cell growth curves with MTT method, cytometric cell cycle analysis with fluorescence activated cell sorter and DMA ladder fragmentation.Results:Part 1: 7 Point and frameshift mutations in the exons 4-7 of p53 gene were identified in 9 keloid tissues and their fibroblasts with PCR-SSCP and DNA sequencing. No p53 gene mutations were detected in normal scar, healthy skin and blood samples of any of the patients.Part 2: The recombinant adenovirus containing human wide-type p53 (Ad-p53) could transfect fibroblasts of keloid in vitro with high transfer rate. The exogenous gene expression appeared at 1d, reached top at 3d and declined gradually.The exogenous p53mRNA and P53 protein were expre...