摘要
多胺是机体中一种重要的阳离子脂肪族有机胺,在细胞增殖、分化中起重要的作用,细胞周期的维持也需要多胺的合成。鸟氨酸脱梭酶(ODC)和S-腺苷甲硫氨酸脱羧酶(AdoMetDC)是多胺生物合成的的关键酶。研究已证实,肺肿瘤中多胺水平明显增高,鸟氨酸脱羧酶(ODC)和S-腺苷甲硫氨酸脱羧酶(AdoMetDC)的表达也明显高于正常肺组织。我们在成功构建并扩增出ODC和AdoMetDC双反义RNA腺病毒载体的基础之上。研究鸟氨酸脱羧酶和S-腺苷甲硫氨酸脱羧酶双反义腺病毒对肺癌增殖和侵袭抑制作用
目的
探讨重组鸟氨酸脱羧酶和S-腺苷甲硫氨酸脱羧酶双反义腺病毒载体(Ad-ODC-AdoMetDCas)介导的鸟氨酸脱羧酶(ODC)和S-腺苷甲硫氨酸脱羧酶(AdoMetDC)反义RNA对肺癌细胞多胺合成、增殖和侵袭的抑制作用。
方法
1.构建并扩增出ODC和SAMDC双反义RNA腺病毒载体;
2.采用活细胞计数法实验观察Ad-ODC-AdoMetDCas对肺癌A-549细胞生长增殖的影响;
3.采用Western印迹和高效液相色谱分别检测腺病毒对肺癌A-549细胞中ODC和AdoMetDC蛋白表达以及胞内多胺含量;
4.采用活细胞计数和流式细胞仪分别检测腺病毒介导的鸟氨酸脱羧酶反义RNA和Ad-ODC-AdoMetDCas对A-549细胞增殖和细胞周期分布的影响;
5.应用Matrigel侵袭实验分析腺病毒对肺癌细胞侵袭活性的改变。
结果
1.成功构建并扩增出ODC和SAMDC双反义RNA腺病毒载体
2.当感染效率为50时,腺病毒对A-549细胞感染率约75%;
3.Ad-ODC-AdoMetDCas可明显抑制A-549细胞ODC和AdoMetDC基因表达;A-549细胞感染Ad-ODC-AdoMetDCas后,胞内多胺含量明显降低;
4.感染Ad-ODC-AdoMetDCas的A-549细胞停滞在G_1期;
5.Ad-ODC-AdoMetDCas可显著降低A-549细胞的体外侵袭能力。
结论
ODC和AdoMetDC双反义腺病毒能够显著抑制肺癌A-549细胞的增殖和侵袭。
The polyamines are positively charged aliphatic amines at physiological.Polyamines are known to be critically involved in cell growth and have been implicated in the process of cell transformation.The level of polymines are high in cancer cells and tissues,and rapid tumor growth has been associated with remarkable elevation of polyamine biosynthesis and accumulation. Decarboxylase(ODC)and S-adenosylmethionine decarboxylase (AdoMetDC),the key enzymes in the biosynthesis of polyamines, are found to increase in cancer cells especially lung cancers.We are studying the inhibitory effects of Ad-ODC-AdoMetDCas on polyamine biosynthesis,lung cancer cell proliferation and invasion on the ground of the successfully constructed and amplified of antisense bicistronic recombinant adenoviruses of ODC and AdoMetDC.
Objective
To study the inhibitory effects of Ad-ODC-AdoMetDCas on polyamine biosynthesis,lung cancer cell proliferation and invasion.
Methods
1.The construction and amplification of antisense bicistronic recombinant adenoviruses of ODe and SAMDC.
2.Adenovirus-mediated gene transduction efficiency was assessed with counting GFP-positive cells using trypan blue.
3.Western Blot and HPLC were used to detect ornithine decarboxylase(ODC)and S-adenosylmethionine decarboxylase (AdoMetDC)expression and polyamine content in A-549 cells respectively.
4.Viable cell counting and cell cycle analysis were adopted to evaluate cell growth and cell cycle distribution.
5.A-549 cell invasion in vitro was detected with Matrigel invasion assay.
Results
1.The antisense bicistronic recombinant adenoviruses of ODC and AdoMetDC are successfully constructed and amplified.
2.Approximate 75%of A-549 cells were infected with Ad-ODC-AdoMetDCas when multiplicity of infection reached 50.
3.Our study demonstrated that Ad-ODC-AdoMetDCas vector-mediated gene transfer inhibited tumor cell growth through the blockade of polyamine synthesis pathway.
4.The tumor cells were arrested at cell cycle G1 phase after gene transfer.
5.Gene transferred tumor cells were shown to possess markedly decreased invasiveness.
Conclusion
Ad-ODC-AdoMetDCas has significant inhibitory effects on lung cancer cell proliferation and invasion and bears therapeutic potential for the treatment of lung cancer.
引文
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