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目的探讨半乳糖受体介导的增殖细胞核抗原(PCNA)反义核酸(ASODN)对肝癌Bel-7402细胞增殖的靶向抑制作用。方法选择来源于3个不同系统的肿瘤细胞:肝癌Bel-7402细胞、肺腺癌GLC-82细胞、慢性髓系白血病K562细胞,将半乳糖-聚乙烯亚胺(Gal-PEI)与PCNA反义核酸结合形成交联复合物Gal-PEI-ASODN,台盼兰拒染计数法观察不同浓度Gal-PEI-ASODN对这3种细胞增殖的影响;流式细胞仪检测3种细胞对羧基荧光素(FAM)标记的Gal-PEI-ASODN的摄取率及细胞内平均荧光强度;相差/荧光显微镜观察FAM标记的Gal-PEI-ASODN及ASODN进入3种不同细胞的情况。结果浓度从10μmol/L到40μmol/L的ASODN作用于Bel-7402细胞48 h后,可抑制其增殖,IC50为29.3μmol/L。浓度从0.05μmol/L到0.25μmol/L的ASODN作用于Bel-7402细胞48 h后,均未显著抑制其增殖;相同浓度的Gal-PEI-ASODN作用于3种不同细胞48 h后,对GLC-82细胞、K562细胞的增殖未产生显著抑制作用,而Bel-7402细胞的增殖却受到明显抑制。对于Bel-7402细胞,Gal-PEI-ASODN的IC50为0.06μmol/L,与单纯ASODN组相比,增敏倍数为488倍。FAM标记的Gal-PEI-ASODN或ASODN与不同细胞孵育0.5 h到2.0 h内,Gal-PEI-ASODN-Bel-7402细胞组所有时间点的细胞荧光摄取率、胞内平均荧光强度均显著高于ASODN-Bel-7402细胞组、Gal-PEI-ASODN-GLC-82细胞组和Gal-PEI-ASODN-K562细胞组。FAM标记的Gal-PEI-ASODN或ASODN与Bel-7402、GLC-82、K562细胞作用1 h后,Gal-PEI-ASODN-Bel-7402组的细胞荧光摄取率较高(反义核酸大量分布于细胞的胞浆内),其他各组细胞荧光摄取率较低。结论半乳糖受体介导的PCNA反义核酸能被肝癌Bel-7402细胞高效地摄取,抑制Bel-7402细胞的增殖,增强反义核酸的作用效果,具有靶向性。
Objective To investigate the inhibitory effect of ASODN mediated by galactose receptor on the proliferation of Bel-7402 hepatocellular carcinoma cells. Methods Tumor cells from three different systems were selected: Bel-7402 hepatocellular carcinoma, GLC-82 lung adenocarcinoma and K562 chronic myeloid leukemia cells. Antisense of Gal-PEI and PCNA PEI-ASODN was detected by trypan blue exclusion-counting method to observe the proliferation of these three kinds of cells with different concentrations of Gal-PEI-ASODN. Flow cytometry was used to detect the effect of Gal-PEI- FAM) labeled Gal-PEI-ASODN uptake rate and intracellular fluorescence intensity; phase contrast / fluorescence microscopy FAM labeled Gal-PEI-ASODN and ASODN into three different cells. Results The ASODN with a concentration of 10μmol / L to 40μmol / L could inhibit the proliferation of Bel-7402 cells for 48 h with IC50 of 29.3μmol / L. ASODN at a concentration of 0.05μmol / L to 0.25μmol / L did not significantly inhibit the proliferation of Bel-7402 cells 48h after treatment with the same concentration of Gal-PEI-ASODN for 48h, -82 cells, the proliferation of K562 cells did not produce significant inhibition, while the proliferation of Bel-7402 cells was significantly inhibited. For Bel-7402 cells, the IC50 of Gal-PEI-ASODN was 0.06 μmol / L, which was 488 fold higher than that of ASODN alone group. The fluorescence uptake rate and intracellular mean fluorescence intensity of Gal-PEI-ASODN-Bel-7402 cells incubated with FAM-labeled Gal-PEI-ASODN or ASODN for 0.5 h to 2.0 h were significantly higher than those of Gal- ASODN-Bel-7402 cells, Gal-PEI-ASODN-GLC-82 cells and Gal-PEI-ASODN-K562 cells. The fluorescence uptake rate of Gal-PEI-ASODN-Bel-7402 group was higher after F-labeled Gal-PEI-ASODN or ASODN was treated with Bel-7402, GLC-82 and K562 cells Cell cytoplasm), the other groups of cells lower fluorescence uptake rate. Conclusion Galactose receptor mediated PCNA antisense nucleic acid can be efficiently taken up by Bel-7402 hepatocellular carcinoma cells, which can inhibit the proliferation of Bel-7402 cells and enhance the effect of antisense oligonucleotides.