PEI/ASON纳米组装体对肺腺癌A549细胞增殖活性及其hTERT基因表达的影响

PEI/ASON-mediated nanoparticle assembly suppresses proliferation and hTERT mRNA expression in lung cancer cell line A549

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 determine the effect of nanoparticles for antisense oligodeoxynucleotide （ASODN） of human telomerase reverse transcriptase （hTERT） mRNA on the proliferation and hTERT expres- sion in A549 cells. Methods MTT assay was used to detect the proliferation in A549 cells, and the cells trea- ted by ASODN, PEI/SODN and PEI/ASODN （40 Ixg/ml）. Intracellular fluorescence intensity was detected by laser confocal microscopy in the cells after being transfected with 5＇-FITC-labelled ASODN （FASODN）. In- verse microscopy was used to observe the modality of A549 cells transfected by PEI/ASODN. The expression of hTERT mRNA were determined by RT-PCR. The expression of hTERT protein was detected by immunohisto- ehemictry assay kit. Cell apoptosis were determined by flow cytometry （FCM）. To observe the tumor targeting effect of NGR （Asn-Gly-Arg） , a total of 36 A549 cells transplanted nude rats were injected with ASON, PEI/ ASON, PEI/ASON-PEG and PEI/ASON-PEG-CNGR solutions respectively via tail vein. The distribution of PEI/ASON-PEG-CNGR in the heart, brain, kidneys, lungs and liver was observed in 1, 3 and 6 h after injec- tion. Results PEI/ASODN exerted inhibitory effects on the proliferation in A549 cells in a time-dependent manner. The intracellular fluorescence in PEI/FASODN treated cells was obviously stronger than that in FASODN group （PEI free ） after transfection. After transfecting PEI/ASODN for 72 h, the hTERT mRNA expression was decreased obviously compared with control group （P 〈 0. 05 ）. The hTERT protein expression of PEI/ASODN group was reduced. FCM showed that the early and later apoptosis rates in PEI/ASODN group was 36. 53% and 54.28% resnaetlve]v_ Animal in ~i~,n aYnorlrn,~nt ~h i-1 fh^t O1~1/A~~31~ DI^C ~~TC^D L.A -~^Atumor targeting effect. Conclusion PEI/FASODN of hTERT can inhibit the proliferation in A549 cells effec- tively, suppress mRNA and protein expression of hTERT and cause cell apoptosis.