人瘢痕组织TIMP-1核酶表达载体的构建及临床意义

The establishment and transcripting of TIMP-1 ribozyme plasmids of human scar tissue in vitro

目的 设计能特异性切割人类瘢痕组织中TIMP - 1mRNA的核酶 ,构建其体外表达载体并大量制备核酶 ,以研究特异性阻断TIMP - 1基因表达对瘢痕胶原降解的影响。方法 根据Symons的“锤头结构” ,以人TIMP - 1的mRNA为靶RNA ,设计核酶 ,以P 1.5为载体 ,制备TIMP - 1特异性核酶的克隆 ,并通过转录制备大量核酶。结果 针对第 12 3 ,2 99和 3 5 3位这三个位点设计了三个核酶 ,合成核酶基因后 ,构建了其表达载体并完成了克隆 ,通过体外转录证实克隆正确。结论 核酶体外表达载体的构建及克隆是研究核酶活性 ,抑制TIMP - 1基因表达 ,调控胶原降解的关键步骤之一。人瘢痕组织TIMP - 1核酶体外表达载体的成功构建及克隆 ,为进一步通过反义抑制 ,阻断人瘢痕组织TIMP - 1过度表达 ,实现对病理性瘢痕胶原代谢的调控 ,奠定了坚实的基础。

Objective To design the ribozymes which may cleave the mRNA of human TIMP-1. Establishing plasmids transcripting the ribozymes to produce abundant ribozymes. Methods The TIMP-1 mRNA was taken as the target RNA. Ribozymes were designed according to the 'hammerhead structure' described by Symons. To establish clones of TIMP-1 ribozymes use P 1.5. Then obtain abundant ribozymes by transcripting. Results Three ribozymes targeting the nt 123, nt 299 and nt 353 on TIMP-1 mRNA were designed. The ribozyme gene has been synthesized, plasmids cloned and abundant ribozymes obtained. Conclusion Computer assisted design is indispensable in studying ribozyme. The establishment of plasmids transcripting ribozymes is the key step of studying their activity.