摘要
目的阿霉素是一种临床上广泛使用的抗肿瘤药物,但其治疗作用的发挥受到耐药性的限制。目前研究显示阿霉素主要通过诱导活性氧自由基的产生而杀死肿瘤细胞。人8-羟基鸟嘌呤-DNA糖苷酶(HOGG1)是一种DNA氧化损伤的修复酶,可特异性的切除由活性氧产生的8-羟基鸟嘌呤。针对这一机制,设计并合成HOGG1特异性的锤头状核酶基因,构建其真核表达载体并初步探讨在该基因作用下,人肺腺癌A549细胞株对阿霉素的药物敏感性的影响。方法人工设计并合成核酶基因(RZ),将其克隆到真核表达载体pcDNA3.1(+)中,阳性重组子由BamH和EcoR酶切,琼脂糖凝胶电泳鉴定及DNA测序证实。大量抽提阳性重组子并在非脂质体转染试剂FuGENE6的介导下引入A549细胞株,RT-PCR(逆转录多聚酶链反应)法鉴定转染,并半定量检测转染细胞中HOGG1mRNA表达的改变。MTT法检测细胞转染前后对阿霉素敏感性的变化;彗星试验检测细胞转染前后DNA氧化损伤修复能力的改变。结果经酶切电泳及DNA测序证实合成的核酶基因序列成功克隆入pcDNA3.1(+)中,命名为pcDNA3.1(+)-RZ。经RT-PCR法鉴定质粒成功导入靶细胞中,并检测到转染核酶的靶细胞中HOGG1mRNA表达下降36%,与空白细胞组比较差异具有统计学意义(P<0.05)。MTT法检测显示转染细胞组对阿霉素的药物敏感性增加,与未转染细胞组之间差异具有统计学意义(P<0.05);彗星试验表明在1.0μg/mL阿霉素作用下,转染细胞组较未转染细胞组DNA损伤程度增加(P<0.05),但无时效关系。结论成功构建HOGG1特异性锤头状核酶真核表达载体。核酶在A549细胞内有效抑制靶基因HOGG1的表达,增加了该细胞对抗肿瘤药物阿霉素的敏感性。为进一步研究HOGG1基因的功能奠定了基础。
Objective Adriamycin is widely used as an effective anti-tumor drug clinically treating a number of human cancers, but the effect of adriamycin is limited by drug resistance. The various kinds of investigations indicated that the anti-tumor activity of adriamycin resulted from drug-induced free radical formation. The free radicals could lead to oxidative DNA damage, and the lesion would be repaired by base excision repair （BEB） pathway. Human 8-oxoguanine DNA glycosylase 1 （HOGG1） is a key enzyme on BER pathway. To study the influence and biological mechanism of the HOGG1 to adriamycin drug-sensitivity, the eukaryotic expression vector with gene of hammerhead ribozyme targeting HOGG1 mRNA would be constructed and identified, and then the change of drug-sensitivity in lung cancer A549 ceils would be investigated. Methods According to computer design, two specific restriction site BamH Ⅰ and EcoR Ⅰ were added to both ends of the ribozyme gene, then the modified ribozyme gene was synthesized and cloned into the eukaryotic expression vector peDNA3.1 （ ＋ ）. The positive recombinants were screened by ampiciilin resistance, and plasmids were extracted from the positive recombinants and digested by BamH Ⅰ and EcoR Ⅰ , and then were analyzed by agarese gel electrophoresis and DNA sequencing. The recombinants were transiently transfected into A549 ceils. The positive recombinants were identified by reverse transcription-polymerase chain reaction （BT-PCB） targeting to NEO gene, which was a neomycin resistance gene for selection of stable ceil lines and only existed in vectors. The changes of HOGG 1 mRNA in A549 cells were detected by RT-PCR. Then the cellular sensitivity to adriamycin was tested by comparison between untransfected cells and transfected ceils by MTr assay. The adriamycin-induced DNA damage was investigated by comet assay or single ceil gel electrophoresis （SCGE） between untransfected and transfected cells. Results The recombinatns containing the ribozyme gene were successfully selected by restriction endonuclease digestion and agarose gel electrophoresis, and were further proved by DNA automatic sequencing. A549 ceils containing the recombinants were identified by RT-PCR, because NEO genes were amplified only in cells transfected successfully. The expression of HOGG1 mBNA in A549 transfected with ribozyme gene was 36% significantly less than in control ceils（ P 〈 0.05）. MTr assay showed that the sensitivity of transfected cells to adriamycin were significantly increased in comparison with untransfected ceils （P 〈 0.05）. The comet assay showed that the extent of DNA damage induced by adriamycin was worse in transfected ceils than unrtransfected cells （ P 〈 0.05）, but there was no time-dependent reaction correlation observed in ceils. Conclusion The eukaryotic expression vector with gene of hammerhead ribozyme targeting HOGG1 mBNA was constructed successfully, and effectively inhibited the expression of HOGGI gene in lung cancer A549 cells, and increased the cellular sensitivity to adriamycin, and will help to study deeply the functions of base excision repair genes-HOGG1.
出处
《四川大学学报（医学版）》
CAS
CSCD
北大核心
2006年第2期165-170,共6页
Journal of Sichuan University(Medical Sciences)
基金
国家自然科学基金(批准号30571535)资助~~
