靶向apollon反义核酸抑制结肠癌细胞增殖并提高化疗药物的敏感性  

Knock-down of apollon gene by antisense oligodeoxynucleotide inhibits the proliferation of Lovo cells and enhances chemo-sensitivity

作　　者：何金花[1,2] 张小鹰[1,3] 吴风云[1,4] 廖晓莉[1] 王威[1] 蒋建伟[1] 

机构地区：[1]暨南大学医学院生物化学教研室,广东广州510630 [2]广东省广州市番禺区中心医院检验科,广东广州511400 [3]广东省佛山市南海区妇幼保健院检验科,广东佛山528200 [4]浙江省温州市平阳县人民医院,浙江温州325400

出　　处：《药学学报》2011年第2期138-145,共8页Acta Pharmaceutica Sinica

基　　金：广东省科技计划项目(2009B080701051);广东省医学科研基金(A2009348)

摘　　要：研究凋亡抑制蛋白(inhibitor of apoptosis proteins,IAPs)家族成员apollon反义核酸(antisense oligodeoxynucleotide,ASODN)作用于结肠癌Lovo细胞,观察其对细胞的增殖抑制作用、致凋亡作用及对某些化疗药物敏感性的影响。将人工合成的apollon ASODN,经脂质体包裹后作用于结肠癌Lovo细胞48 h后,采用WST法与克隆形成抑制实验检测不同浓度的apollon ASODN对Lovo细胞增殖抑制作用;实时荧光定量RT-PCR检测细胞apollon mRNA的表达水平;流式细胞术检测细胞周期和细胞凋亡;Hoechst 33258染色观察Lovo细胞凋亡的形态学改变;采用apollon ASODN联合5-氟尿嘧啶(5-FU)、顺铂(DDP)、盐酸表柔比星(EPI),观察对Lovo细胞增殖抑制作用。实验表明,apollon ASODN转染Lovo细胞48 h,能明显下调apollon mRNA的表达,细胞增殖和克隆形成均被显著抑制(P<0.05),并呈浓度依赖关系。Apollon ASODN组G0/G1期细胞减少,S期细胞增多,出现明显的S期阻滞。流式细胞术检测结果显示,apollon ASODN组存在明显的细胞凋亡,经荧光显微镜观察,可见核固缩、边聚、裂解等细胞凋亡形态学变化。0.08μmol.L-1 apollon ASODN与不同浓度的化疗药物(5-FU、DDP、EPI)联合作用于Lovo细胞48 h,可提高Lovo细胞对这些化疗药物的敏感性,增敏倍数分别为2.58、4.47和5.33倍。结果提示,apollon ASODN可下调apollon基因的mRNA表达水平,抑制Lovo细胞增殖,诱导细胞凋亡,使细胞周期阻滞于S期,并提高结肠癌细胞对5-FU、DDP、EPI的敏感性。In this study,the effects of apollon antisense oligodeoxynucleotide（ASODN） on the proliferation and apoptosis of human Lovo cells in vitro were investigated.Apollon ASODN was incubated with human colorectal Lovo cells for 48 h,the proliferation inhibition and the clone forming rates were detected by WST method and clone formation assay,respectively.The expression of apollon mRNA was analyzed by real time fluorescent quantitative reverse transcription polymerase chain reaction.The percentage of apoptotic cells and cell cycle distribution were determined by flow cytometry.The morphology of apoptotic cells was examined by fluorescence microscope.Lovo cells incubated with apollon ASODN combined with 5-fluorouracil（5-FU）,cisplatin（DDP） or epirubicin（EPI） of different concentrations,cell proliferation inhibition rates were detected with WST method and IC50 was calculated.It was found that ASODN targeting apollon gene could all suppress the growth of Lovo cells and induce apoptosis of these cells significantly（P0.05）.After Lovo cells treated with apollon ASODN for 48 hours,the expression of the apollon mRNA level was suppressed significantly.And a marked concentration-dependent decline of cell proliferation and clone forming,increasing of cell apoptosis levels were observed.The percentage of G0/G1 phage cells was abated and that of S phage cells was increased and the Lovo cells arrested at S phage of the cell cycle detected with flow cytometry.Many Lovo cells stained with Hoechst 33258 exhibited apoptotic morphology such as cell shrinkage,nuclear condensation and nuclear fragmentation.Cell proliferation inhibition was detected and their chemo-therapeutic effects of 5-FU,DDP and EPI on Lovo cells combined with apollon ASODN（0.08 μmol·L-1） were enhanced independently compared with single 5-FU,DDP and EPI groups,and the sensitivity enhanced about 2.58,4.47,and 5.33 times respectively.It can be concluded that ASODN targeting apollon can suppress the expression of apollon mRNA,and inhibit the prolif

关 键 词：APOLLON 反义核酸 细胞凋亡 LOVO细胞 结肠癌 

分 类 号：R963[医药卫生—微生物与生化药学]