摘要
背景与目的:醛糖还原酶相似基因1(aldose-reductaselikegene-1,ARL-1)在原发性肝癌中高表达,与肝癌细胞对化疗药物耐药有关。本研究拟建立转染ARL-1的人肝癌细胞株,观察转染ARL-1的HepG2细胞对含醛基抗肿瘤药物耐药性的改变,利用基因表达谱芯片筛选HepG2细胞转染ARL-1后差异表达耐药相关基因。方法:采用脂质体转染PBK/ARL-1质粒到HepG2细胞,获得高表达ARL-1的单克隆细胞株;RT-PCR、流式免疫荧光和免疫组化鉴定高表达ARL-1的HepG2细胞;应用MTT法和细胞凋亡分析研究HepG2细胞和转染ARL-1的HepG2细胞对含醛基抗肿瘤药物阿霉素(ADM)和丝裂霉素(MMC)的耐药性,以5-氟尿嘧啶(5-FU)(不含醛基)为对照;将Cy3和Cy5两种荧光染料分别标记两种细胞的cDNA探针,与人肝癌基因表达谱芯片进行杂交与扫描,观察转染ARL-1基因HepG2与对照组HepG2细胞在基因表达谱方面的差异,筛选耐药相关基因,并运用RT-PCR和Westernblot对部分差异表达相关基因进行初步证实。结果:与对照组HepG2细胞相比,转染ARL-1基因HepG2细胞高表达ARL-1蛋白,明显增强对含醛基的抗肿瘤药物如ADM、MMC的耐药性,分别提高2.6倍和3.47倍,用5-FU处理两组细胞,则未发现有明显的耐药差异(ADM组t=6.39,P<0.05;MMC组t=30.06,P<0.01;5-FU组t=0.684,P>0.05);芯片扫描结?
BACKGROUND &OBJECTIVE: Aldose reductase like gene-1 overexpres se s in hepatocellular carcinomas; it might be the drug resistance-associated gene to anti-tumor drugs containing carbonyl groups on hepatocellular carcinomas. T his study was designed to establish human hepatocellular carcinoma cell line (He pG2) transfected with aldose reductase like gene-1 (ARL-1),then to observe the changes of drug resistance to anti-tumor drugs with carbonyl group and screen the drug resistance-associated genes through cDNA chip. METHODS: PBK/ARL-1 pla smid was transfected to HepG2 cells by liposome to establish HepG2 monoclonal ce lls with high expression of ARL-1. The HepG2 cells with high expression of ARL -1 were determined by reverse transcription-polymerase chain reaction (RT-PCR ),flow cytometric analysis (FCA), and immunohistochemical staining. MTT assay an d cell apoptosis analysis were performed to determine the drug resistance abilit y of the cells to Adriamycin (ADM) and mytomycin (MMC), which contain carbonyl g roup. 5-fluorouracil (5-FU) that has no carbonyl group was used as control. Th en two kinds of cDNA probes were made from HepG2 cells and ARL-HepG2 cells and labeled with Cy3 and Cy5 dyes. The probes were hybridized to the human liver cDN A chip and differentially expressed genes from the two cells were screened. The genes could be involved in drug resistance were confirmed by RT-PCR and Western blot. RESULTS: Compared with HepG2 cells, HepG2 cells transfected with ARL-1 g ene have an increase in expression of ARL-1. Drug resistance ability of HepG2 c ells transfected with ARL-1 gene to ADM and MMC increased 2.6 and 3.47 folds, r espectively (t=6.39,P< 0.05 in ADM group;t=30.06,P< 0.01 in MMC group). Drug res istance to 5-FU had no statistical difference between them (t=0.684,P >0.05 in 5-FU group). 15 genes down-regulated and 9 genes up-regulated after transfect ed with ARL-1 gene were found by cDNA chip scanned. Some differentially display ed genes such as ubiquitin and MDR confirmed by RT-PCR and Western blot were co nsistent to the results of cDNA chip. CONCLUSION: The HepG2 cells with high expr ession of ARL-1 have more drug resistance to anti-tumor drugs with carbonyl gr oup.Up-regulated MDR expression and down-regulated ubiquitin expression may co ntribute to the drug resistance of HepG2 cells.
出处
《癌症》
SCIE
CAS
CSCD
北大核心
2003年第12期1289-1295,共7页
Chinese Journal of Cancer
基金
国家自然科学基金项目(No.39770863)
关键词
转ARL-1基因
HEPG2
相关基因
筛选
耐药性
肝癌
癌细胞
cDNA chip
Aldose reductase like gene-1(ARL-1)
Hepatocellular car cinomas (HCC)
Drug resistance-associated genes
