脱氧核酶对基质金属蛋白酶-9mRNA地体外剪切-骨肉瘤基因治疗地新策略.docx

王浩的硕士论文 脱氧核酶对基质金属蛋白酶-9mRNA的体外剪切 osteosarcoma relates to a lot of cytokine；of that MMP一9 has important work in the course and relates to prognosis． Deoxyribozymes is a kind of monochain DNA fragment own catalysis fimction：high catalysis activity and structure identification ability．Hitherto many Deoxyribozymes seven categories according to their function had been found and can be divided into the most special one posses RNA cleave activity and can cleave RNA definite spot SO that inactivate gene at mRNA level to adjust protein expression．It will be a new type of gene therapy drug and new type of nucleic tool enzyme apply to investigation of anti-virus，anti—tumor Thus we design and synthesis the special Deoxyribozymes aimed at the MMP．9 mRNA，try to adopt the Deoxyribozymes technique to anti MMP一9 expression SO to fend out a new pathway of gene therapy on osteosarcoma． PART 1：DETECTION THE EXPRESSION LEVEL AND SUBCLONE OF MMP．90F HUMAN OSTEOSARCOMA CELL LINES U-20S， AIM：Detect the expression of MMP一9 in human osteosarcoma cell line U．20S，try to find the action of MMP-9 in growing and transferring of osteosarcoma cell．To produce recombinant expression vector of MMP-9 as the templat of transcription in vitro． METHODS：Cell culturing of U-20S，prepare total RNA and reverse V 王浩的硕士论文 脱氧核酶对基质金属蛋白酶一9mRNA的体外剪切 tasnscript to cDNA as template．PCR the highly conserved gene fragment of MMP一9 and subclone to prokaryotic expression vector PCDNA3．To get a recombinant plasmid named MMP-9——PCDNA3．After transforming the recombinant plasmid into E．coli and screening with antibiotic we got several positive samples．One of them was chosen for sequencing．Finally it can be templat of trancription in vitro after worked by restriction enzyme EcoR I． RESULT：Recombinant vector MMP．．9．．PCDNA3 is successfully constructed． CONCLUSION：Recombinant vector MMP．-9--PCDNA3 is successfully constructed． PART 2 SYNTHESIS THE SPECIFIC DEOXYRIBOZYME TO C