The In Vivo Study With RNAi Against Heat Shock Protein 47 To Collagen Of Pathological Scar

Pathological scars including hypertrophic scar and keloid , usually arise from various skin wounds. It affects not only surface but also function of the patients. It has become a important project to alleviate pathological scars for the kingdom of burn or plastic surgery.The therapeutic methods to pathological scars are limited so far. With rapid development of the techniques of biochemical research and genetic engineering, gene therapy has become a focus project. Gene therapy method gives new hope to cure pathological scars ,although still in experiment stage now.It has been shown in past investigations, that the increasing production and deposition of collagen is the important mechanisms of pathological scars and the increasing is parallel to the upregulation of HSP47 gene. We designed and built HSP47 siRNA plasmid. After the study of cell stage, we built the model of nude mice with hypertrophic scar and keloid from clinical patients. After in vivo interfering experiment in K and HS nude mice models, we hope that RNAi can suppress the expression of HSP47 gene and than reduce the production of collagen . Explore a new and valid method to cure pathological scars. This study includes three parts as follow:1. Build the model of nude mice with hypertrophic scar and keloid Object: We built the model of nude mice with pathological scars to prepare for in vivo interfering of siRNA , while to identify the feasibility and reliability of the model in the morphology and the histology.Method: Pieces of hypertrophic scars and keloids were implanted intosubcutaneous tissue of the nude mice. The mice carried them for a period of23 days . Than these specimens were compared with the original ones underthe light Microscope.Rusults: All mice survived after the surgery . No cell degeneration . Thecollagen pattern of all hypertrophic scars and keloids were remained in everycases.Conclusion: This procedure showed that the viability and morphology ofhypertrophic scars and keliods are sustained when they are implanted in nudemice. It is feasible and reliable to use nude mice as the model of the animalwith hypertrophic scars and keliods.2. Designed and built HSP47 siRNA plasmid .Distill abundance plasmid. Produced the mixture of plasmid and liposome.Object: Designed and selected HSP47 siRNA . Built and purefied plasmid adequate for in vivoMethod: We designed and built HSP47 siRNA plasmids, than insert it incolony DH5a. 3.Purified plasmid using the endofree plasmid maxikit(QIAGEN).Produced the mixture of plasmid and liposome(INVITROGEN 200)Results: The yields was 180ug plasmid per QIAGEN culture volumes in ourexperiment .Our harvest was adequate for next in vivo interfering . All aboveshowed that plasmid building was successful.Conclusion: Built and purified HSP47 siRNA plasmid successfully.3. The in vivo study with RNAi against Heat shock protein 47 toPathological Scar in nude mice modelObject: Investigate the affection of the pathological scars after suppressingthe expression of HSP47 gene with RNAi in nude mice modelMethod: Mixed plasmid that had been purified including HSP47 siRNA andliposome (INVITROGEN 200) in rateably. We injected RNAi mixture intothe scars of nude mice model in experiment group and PBS water(0.25ml)into contrast group at the 16th days after building the models. Afterinterference we observed the specimens and harvested specimens at 7th daysfor biochemical and pathological analysis.Results: We suppress the expression of HSP47 gene and than reduce theproduction of collagen after in vivo interfering experiment in Keloid,but wedid not get the same result in hypertrophic. Conclusion: We can suppress the expression of HSP47 gene and than reduce the production of collagen after in vivo interfering experiment with HSP47siRNA in Keloid nude mice models using RNAi technique. But we did not get the same result in hypertrophic. The succession of our study definituded the mechanism that HSP47 promoting the keloid formation .The result provided a new target to treat keloid and provided us hard-won data too.