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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。
To study the effect of apollon antisense oligodeoxynucleotide (ASODN) on the proliferation of Lovo cells in colon cancer cells and to observe its inhibitory effect on cell proliferation, apoptosis and some chemotherapies The impact of drug sensitivity. Synthetic apollon ASODN was encapsulated in liposome and then treated with Lovo cells for 48 h. The inhibitory effect of different concentrations of apollon ASODN on the proliferation of Lovo cells was detected by WST assay and clonogenic assay. Real-time fluorescence quantitative RT-PCR The expression of apollon mRNA was detected by flow cytometry. The cell cycle and apoptosis were detected by flow cytometry. Morphological changes of apoptosis were observed by Hoechst 33258 staining. 5-fluorouracil (5-FU) and cisplatin (DDP) ), Epirubicin hydrochloride (EPI), to observe the inhibition of proliferation of Lovo cells. Experiments show that apollon ASODN transfected Lovo cells for 48 h, can significantly down-regulate apollon mRNA expression, cell proliferation and colony formation was significantly inhibited (P <0.05), and in a concentration-dependent manner. Apollon ASODN group G0 / G1 phase cells, S phase cells increased, a significant S phase arrest. Flow cytometry results showed that apollon ASODN group had obvious apoptosis. The morphological changes of apoptotic cells such as pyknosis, edge aggregation and lysis were observed by fluorescence microscopy. The combination of 0.08μmol.L-1 apollon ASODN with different concentrations of chemotherapy drugs (5-FU, DDP, EPI) in Lovo cells for 48 h increased the sensitivity of Lovo cells to these chemotherapeutic drugs with 2.58, 4.47 and 5.33 times. The results suggest that apollon ASODN can down-regulate the mRNA expression of apollon gene, inhibit Lovo cell proliferation, induce apoptosis, arrest the cell cycle in S phase and increase the sensitivity of colon cancer cells to 5-FU, DDP and EPI.