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目的以聚乙烯亚胺多聚阳离子纳米载体(polyethylenimine,PEI)作为端粒酶逆转录酶(human telomerasereverse transcriptase,hTERT)反义寡核苷酸(antisense oligodeoxynucleotide,ASODN)载体转染A549细胞,研究其对A549细胞的增殖活性及hTERT mRNA表达的影响。方法采用MTT法检测PEI/ASODN纳米组装体对细胞的增殖作用情况;激光共聚焦显微镜检测5’-FITC标记的ASODN(FASODN)转染细胞后PEI/FASODN纳米组装体的细胞摄入情况;RT-PCR检测转染后hTERT mRNA的表达;免疫细胞化学技术检测端粒酶hTERT蛋白的表达;流式细胞术Annexin-V-FITC和PI双标法检测细胞凋亡情况;PEI/ASON-PEG-CNGR组装体动物体内分布实验观察NGR的肿瘤靶向作用。结果 PEI/ASODN纳米组装体转染细胞后产生良好的生长抑制作用;FASODN转染细胞后,PEI/FASODN组细胞内有大量荧光物质,而单纯FASODN组细胞内无明显荧光;RT-PCR检测结果表明PEI/ASODN纳米组装体作用于A549细胞72 h时,hTERTmRNA水平明显降低,与空白对照组相比有显著差异(P<0.05);细胞hTERT蛋白的表达也显著降低;转染72 h后PEI/ASODN组出现明显早期凋亡和晚期凋亡,凋亡率分别为36.53%和54.28%;动物体内分布实验表明PEI/ASON-PEG-CNGR具有良好的肿瘤靶向作用。结论 PEI介导的hTERT ASODN能有效抑制A549细胞增殖,降低hTERT mRNA水平从而抑制hTERT蛋白的表达,使细胞产生凋亡,对该细胞生长有明显抑制作用。
OBJECTIVE To investigate the effect of polyethylenimine (PEI) on A549 cells transfected with human telomerase reverse transcriptase (hTERT) antisense oligodeoxynucleotide (ASODN) On A549 cell proliferation activity and hTERT mRNA expression. Methods MTT assay was used to detect the proliferation of PEI / ASODN nanocomposites. Laser confocal microscopy was used to detect the cellular uptake of PEI / FASODN nanocomposites after transfection of ASODN (FASODN) cells transfected with 5’-FITC. RT The expression of telomerase hTERT protein was detected by immunocytochemistry, the apoptosis was detected by flow cytometry Annexin-V-FITC and PI double labeling method, the expression of PEI / ASON-PEG- CNGR assembly animals in vivo experiment to observe the role of NGR in tumor targeting. Results After transfected with PEI / ASODN nanocomposites, there was a good growth inhibition effect. After transfected with FASODN, there were a large number of fluorescent substances in the cells of PEI / FASODN group, but no obvious fluorescence in the cells of FASODN group. The results of RT-PCR The results showed that the expression of hTERT mRNA in A549 cells was significantly decreased after exposure to PEI / ASODN nanocomposites for 72 h (P <0.05), and the expression of hTERT protein was also significantly decreased after 72 h of transfection / ASODN group showed obvious early apoptosis and late apoptosis with the apoptotic rates of 36.53% and 54.28%, respectively. The in vivo distribution experiments showed that PEI / ASON-PEG-CNGR had good tumor targeting effect. Conclusions PEI-mediated hTERT ASODN can effectively inhibit the proliferation of A549 cells, decrease the expression of hTERT mRNA, and inhibit the expression of hTERT protein, resulting in the apoptosis of cells and significantly inhibiting the growth of A549 cells.