摘要
背景与目的:核苷酸切除修复机制是细胞修复损伤DNA的重要途径,肿瘤细胞的耐药常伴随DNA损伤修复基因表达增强,采取反义策略降低细胞的DNA损伤修复能力可以增加肿瘤细胞的药物敏感性。本研究拟构建能在哺乳动物细胞中表达XPB反义RNA的表达质粒pcDNA-XPB/AS(XPB:着色性干皮病B基因),并初步探讨其对肺癌细胞DNA损伤修复能力及抗肿瘤药物耐药性的影响。方法:采用RT-PCR技术扩增的XPBcDNA5'端一段69～520bp的序列被反向插入表达质粒pcDNA3.1/His。将该重组质粒瞬时转染肺癌A549细胞。单细胞凝胶电泳(SCGE)比较阿霉素诱导的转染前后细胞DNA损伤修复情况。MTT法检测转染前后细胞对阿霉素的敏感性。结果:酶切图谱分析和基因测序证实反义表达质粒构建成功。RT-PCR显示转染细胞XPBmRNA表达水平下降。以4.0μg/ml阿霉素诱导细胞DNA损伤,SCGE显示转染细胞修复DNA损伤能力受到抑制。MTT显示未转染细胞与转染细胞对阿霉素的敏感性存在差别,但无统计学意义。结论:构建的反义表达质粒能下调转染细胞XPBmRNA表达,抑制细胞DNA损伤修复能力,为进一步研究XPB基因功能奠定了基础。
BACKGROUND &OBJECTIVE:Nucleotide excision repair is an important pathway for cellular DNA d amag e repair.The drug resistance of tumor cell is often companied with the enha nced expression of DNA repair g enes.Down-reg ulation of DNA repair capac ity by antisense strateg y can increase the drug sensitivity of tumor cells.The aim of this study was to construct the eukaryotic expression plasmid pcDN A-XPB /AS(XPB:xeroderma pig mentosum B)and to investig ate the function of XP B g ene and its roles in chemotherapeutic drug sensitivity in lung cancer A549cell.METHODS :The XPB cDNA(69-520bp)frag ment amplified by reverse trans cription polymerase chain reaction(RT-PCR)was inserted into pcDNA3.1/His plas mid with an inverted orientation.The recombinant plasmid was transie ntly transfected into A549cells.Th e Adriamycin-induced DNA damag e was c ompared between the transfected and the untransfected cells by sing le cell g el electrophoresis assay(SCGE).The cellular sensitivity to Adriamycin of the transfected and the untransfe cted cells was determined by MTT assay.RESULTS :The successful construction of antisense plasmid was proved by rest riction map and sequence analysis.RT-PCR results showed that the XPB mRNA expression was inhibited in transfected A549cells.SCGE showed that the cellular damag e repair abil ity induced by 4.0μg /ml Adriamycin was suppressed in tr ansfected cells.MTT assay showed the sensitivity of the t ransfected cells to Adriamycin was d ifferent from the untransfected cells but wit hout statistical meaning .CONCLUSION:The antisense plasmid constructed by th e authors can down-reg ulate the expr ession of XPB mRNA in the transfected cells a nd inhibit the cellular DNA damag e re pair ability,providing a basis to furthe r study the g ene function of XPB.
出处
《癌症》
SCIE
CAS
CSCD
北大核心
2003年第9期907-911,共5页
Chinese Journal of Cancer
基金
国家自然科学基金重大资助项目(No.39990570)
