针对点突变癌基因转录物的核酶细胞内性质的研究  

Ribozyme Targeted the Point-Mutation of Activated Oncogene Inhibits Its Expression in vivo

作　　者：刘戈[1] 陈雅文[1] 赵西林[1] 董元舒[1] 陈德风[1] 

机构地区：[1]南开大学分子生物学研究所

出　　处：《Acta Genetica Sinica》1996年第6期477-485,共9页

摘　　要：前文［１］已证实在体外（ｉｎｖｉｔｒｏ）实验接近生理环境的条件下，本室设计、合成并克隆到的核酶能够高效选择性的定点切割Ｔ２４－ｒａｓ活化癌基因转录物。在此基础上，为阐明该核酶在体内（ｉｎｖｉｖｏ）的生理活性，本文又进一步把核酶基因片段克隆在真核表达质粒ｐＳＭＧ上，并将重组质粒以磷酸钙沉淀法转染由Ｔ２４－ｒａｓ基因诱导的转化细胞系。在细胞和分子水平上检测了核酶在真核细胞内的生物学活性：表现为各恶性转化细胞系的形态特征逆转，生长速度减慢，并呈现出重叠生长减弱恢复接触抑制的趋势，细胞凝集行为接近正常、软琼脂集落形成能力下降；同时，引物延伸实验结果也表明：在体内实验条件下，核酶能够特异性切割点突变Ｔ２４－ｒａｓ癌基因转录物，抑制癌变细胞的恶性行为，使其得到一定程度的逆转。We demonstrated previously that the hammerhead ribozyme designed by us against the activated oncogene T24-ras cleaved the target mRNA specifically and efficiently in vitro[1].To study its properties in vivo, we cloned the DNA fragment encoding the ribozyme into the eukaryotic expression vector pSMG and transfected it into the T24-ras gene transformed NIH3T3 cell lines. The intracellular cleavage of 724-ras mRNA was evaluated on either the cytological level or the molecular level. The malignant cells expressing ribozyme were partially reversed in morphology as evidenced by slower growth speed, partial recovery of contact-inhibition, reduced frequency of colony forming in soft agar and close-to-normal behaviour in agglutination teat. The primer extention experiment verified that the ribozyme had efficiently cleaved the transcripts of T24-ras gene at the target site in vivo.

关 键 词：核酶 T24-ras基因 体内活性 癌基因 转录物 

分 类 号：Q550.2[生物学—生物化学]