修饰的反义寡脱氧核苷酸对胰腺癌细胞生长和靶基因表达的抑制作用

Inhibition of cell growth and target gene espression of human pancreatic carcinoma cells by modified antisense oligodeoxynucleotide

目的观察修饰过的反义寡脱氧核苷酸（ＡＳＯＮＤ）对人胰腺癌细胞生长和靶基因表达的抑制作用。方法用人工合成与ｃ－ｍｙｃ和Ｋｉ－ｒａｓ帽区互补的修饰的反义寡脱氧核苷酸，即反义硫代正磷酸寡脱氧核苷酸（ａｎｔｉｓｅｎｓｅｐｈｏｓｏｐｈｏｒｏｔｈｉｏａｔｅｏｌｉｇｏｄｅｏｘｙｎｕｃｌｅｏｔｉｄｅ，ＡＳＰＯＤＮ）处理人胰腺癌细胞系ＰＣ－２和ＰＣ－３。多次小剂量（１０μｇ）或一次性剂量（１５μｇ）ＡＳＰＯＤＮ处理细胞后计数细胞生长率和３Ｈ掺入率，并用逆转录聚合酶链反应检测靶基因表达。结果多次小剂量ＡＳＰＯＤＮ处理后，细胞生长、３Ｈ掺入和靶基因表达的抑制可长达２周以上，到第１４天时实验组的细胞生长率仅为对照组的３８％～４３％，３Ｈ掺入率为对照组的１８％～３３％；靶基因表达明显受抑或下调，经一次性１５μｇＡＳＰＯＤＮ处理后，细胞生长、３Ｈ掺入和靶基因表达的抑制可达４天。结论与以往的研究比较，表明ＡＳＰＯＤＮ较ＡＳＯＤＮ有更强的抑制细胞生长、３Ｈ掺入、靶基因表达的作用。

Objective To investigate the inhibitory effects of modified antisense oligodeoxynucleotide on cell growth, 3H-TdR incorporation rate and target gene expressions of human pancreatic carcinoma cells as a comparison with the effectiveness of nonmodified antisense oligodeoxynucleotide(ASODN)resported previously.Methods Synthesized modified antigsense oligodeoxynucleotides(antisense phosphorothioate oligodeoxynucleotides,ASPODN)complementary to the cap regions of c myc and Ki ras genes were used to treat PC 2 and PC 3 human pancreatic carcinoma cell line cells with multiple small(10 μg)doses or one single dose(15 μg).After treatment,cell growth rates and 3H TdR incorporation rates were estimated,and the concurrent oncogene expressions were studied by adopting RT PCR technique.Results After multiple ASPODN exposures,the cell growth rates and 3H--TdR incorporation rates were significantly inhibited,the inhibition was maintained for more than two weeks.On the 14 thday,the cell growth rates of the ASPODN groups were reduced to 38%～43% of that of the controls,and the 3H TdR incorporation rates were 18%～33% of that of the controls,there were also marked inhibition or down regulation of target genes(c myc and Ki ras)expressions.After the 15 μg single dose treatment,the inhibition of cell growth, 3H TdR incorporation and target gene expressions lasted for 4 days.Conclusions The results of this study confirm the fact that ASPODN exerts a more strong inhibitory effect on pancreatic carcinoma cells than the non modified ASODN. Key wotds Pancreatic neoplasms Oligonucleotides,antisense PolymerasechainreactionProto oncogenes1Mutation and methylation of CDKN2 gene in human head and necd carcinomas.$$$$ Tao Jiangchuan,Wu Bingquan,Facg Weigang,et al.Department of Pathology,Neijing Medical University,Beijing 100083 Abxtract Objective To study mutations of CDKN2 gene in human head and neck carcinom as(HNC)and to elucidate the role of this gene in tumorigenesis. Methods PCR SSCP and sequencingtechniques were used to detect mutations in fresh specimens from 36 HNC cases.Specimens from 20 cases were further analysed for methlyation status of the CpG islands in the first exon of the gene using restriction enzyme digestion plus Southern blotting analysis. Results CDKN2 gene mutations were detected in 4 HNC cases of which 2 were missense mutation and the other 2 were frameshift mutation.Methylation status analysis revealed that 8 out of 20 HNC cases had de novo methylation of CpG isla nd within the first exon of CDKN2 gene.Conclusion The results shown here suggest that in addition to gene mutation and deletion,de novo methylation of the CpG island may be a frequent event in human carcinogenesis.