【摘要】 本文采用逆转录－聚合酶链式反应（ＲＴ－ＰＣＲ），并结合ＰＣＲ产物直接测序法，证明人大肠癌细胞系ＨＲ８３４８和ＨＣｅ８６９３的ｋ－ｒａｓｃＤＮＡ片段（第３～８３密码子）中存在第１２密码子ＧＧＴ→ＧＡＴ突变，未发现其它突变或ＲＮＡ剪接异常．用分别与野生型和上述突变型ｋ－ｒａｓ第９～１５密码子互补的１８聚硫代磷酸型寡核苷酸（两者仅存在一个碱基差别）及随机设计的对照寡核苷酸（终浓度为６μｍｏｌ·Ｌ－１），发现与突变型ｋ－ｒａｓ互补的寡核苷酸能抑制ＨＣｅ８６９３细胞生长并有ｐ２１ｒａｓ蛋白表达量的抑制；而与野生型ｋ－ｒａｓ互补的寡核苷酸及随机合成的对照寡核苷酸对之无明显抑制作用，说明ｃ－ｋ－ｒａｓ反义寡核苷酸对ＨＣｅ８６９３细胞生长和ｐ２１ｒａｓ蛋白合成的抑制作用存在明显的核苷酸序列特异性．更多还原

【Abstract】 Only one base substitution in codon 12 (GGT to GAT) of the k-ras cDNA fragment (242 bp, codon 3-83) in human colorectal cancer cell lines HR8348 and HCe8693 was demonstrated by PCR direct sequencing in combination with reverse-transcription polymerase chain reaction (RT-PCR). No base change at other codons and no mRNA splicing abnormality were found. 18-mer phosphorothioate oligonucleotides which targeted to codon 9-15 of wild-type and mutated k-ras (only one base difference) or random oligos were synthesized respectively. At final concentration of 6 μmol·L -1 , it was found that antisense oligonucleotides which complement to mutated k-ras gene inhibited HCe8693 cell growth and p21 ras expression, in contrast, no effects were found to those which complement to wild-type k-ras gene and random oligos. These results suggested that c-k-ras antisense oligonucleotides may have a sequence-specific inhibitory effect on cell growth and p21 ras protein synthesis in HCe8693 cells.更多还原