Study On The In Vitro Cleavage Activity Of The Second Exon Ribozyme Of Type ¢ñ And ¢ó Procollagen Gene On Target Rna
Keywords
Procollagen Gene, Ribozyme, In Vitro Activity, Fibroblasts, Scar
Abstract
Objective To study the cleavage activity and reaction conditions of hammerhead ¦Á1 type I and type III procollagen gene exon 2 fragment ribozymes in vitro on their respective target RNA molecules. The effects of two antisense ribozymes on collagen synthesis by fibroblasts in scars were also observed. Methods The recombinant plasmids (pt-¢¡, pt-¢£) containing exon 2 fragments of ¦Á1 type I and type III procollagen genes were transcribed with 32p label in vitro to form the product target RNA. At the same time, the recombinant plasmids (pt-g¢¡, pt-g¢£) containing specific ribozyme genes were subjected to non-labeled in vitro transcription. The products (ribozymes) were mixed and reacted with their respective 32p-target RNA according to different conditions, and then radiated after electrophoresis. Develop and observe the results. The constructed ribozyme was packaged with liposomes and introduced into cultured fibroblasts, and the effect of the ribozyme on the synthesis of type I and type III collagen mRNA in fibroblasts was observed using image analysis. Results Both ribozymes can effectively cleave their respective target RNAs at 37¡ãC and 42¡ãC, and have a wide range of mg2+ concentration requirements (10~20mmol/l); the reaction temperature gradually dropped from 65¡ãC to 37¡ãC and was maintained at 37¡ãC. Under these conditions, the ribozyme cleavage activity was significantly improved. The expression of type I and III collagen mRNA was significantly reduced, collagen production was reduced, and collagen production was significantly inhibited. Conclusion The ribozyme targeting the exon 2 fragment of ¦Á1 type I and type III procollagen genes can effectively cleave target RNA in vitro and effectively inhibit the synthesis of collagen in fibroblasts in scars.
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Original research was done by Zhu Jiayuan, Zhu Bin, Xu Bing, Zhang Tao, Chen Dong, Tang Bing, Li Xinqiang
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