mRNA差异显示法筛选卵巢癌细胞株细胞耐药相关基因的初步研究

Screening of drug resistance related genes from human ovarian cancer cell line OC3/Adr by DD PCR

研究卵巢癌耐药细胞系OC3/Adr和卵巢癌敏感细胞系OC3在mRNA分子水平上的表达差异。方法 :用高浓度阿霉素间歇诱导法建立卵巢癌耐药细胞系 ;应用mRNA差异显示方法找出两株细胞间cDNA差异表达片段 ;RT PCR检测耐药细胞表达差异片段在卵巢癌细胞和组织中的表达。结果 :建立了稳定传代的卵巢癌耐药细胞系OC3/Adr,耐药指数为15 4;OC3/Adr对VP16,CDDP,5 FU的耐药指数分别11 6,10 5,8 8 ,与亲本细胞相比 ,OC3/Adr细胞形态和周期发生变化。筛选到差异表达片段数十条,对其中的四个片段进行了序列测定和同源性分析 ,S1与精子蛋白高度同源;S2与已定位人18号染色体一段序列高度同源 ,但功能未知 ;S3、S4为未知序列 ,S2在耐药细胞中特异表达 ;S3在卵巢癌与正常卵巢、卵巢良性肿瘤中表达有明显差异。结论 :阿霉素诱导的细胞耐药现象与多基因参与有关 。

Objective:To screen novel genes related to adriamycin (Adr) resistance from human ovarian cancer resistance cell line OC3/Adr Methods: Multidrug resistant ovarian cancer cell line OC3/Adr was induced by intermittent treatment of the human parent cell line OC3 with high concentration Adr The difference of genes expression was screened by using differential display analysis to the acquired Adr resistance subline OC3/Adr and its parent cell line OC3 Results:OC3/Adr cell line was obtained which appeared more resistance to Adr than the parent cell line OC3 and with the resistance index(RI) of 15 4 The OC3/Adr cell line also showed cross resistance to other anti cancer drugs(VP16,CDDP,5FU), grew slowly and exhibited changes of cell cycle A number of differentially expressed ESTs (Expressed Sequence Tags,ESTs) were identified at mRNA level between the OC3/Adr and OC3 Four of 18 different ESTs were sequenced The 431/432 base pair S1 was homologous of human sperm zona pellucida binding protein,while the other two ESTs ,S3 and S4, were new gene segments,which were registered to GenBank with the number of AF 117656 and AF 126507 respectively Particularly,the expression of S2 sequence increased in the all drug resistance cell lines and S3 sequence overexpressed in human ovarian cancer tissues as compared with benign ovarian tumors Conclusion:Drug resistance induced by Adr in orarian cancer OC3/Adr is involved with changes of multiple gene expressions