Tet-on系统诱导esp1基因表达对A_(549)细胞染色体的影响

Effect of conditional expression of esp1 gene by Tet-on system on ploidy in A_549

目的观察肺腺癌Am细胞经Tet-on系统诱导表达espl基因后其染色体数目的改变。方法利用PTet—on基因表达调控系统，先后将调控质粒Ptet-on和反应质粒PTK-Hyg与PTRE-EGFP-espl以合适的比例用FuGENE6脂质体共转染入A-细胞，经G418和潮霉素筛选成活并带有绿色荧光的细胞为转染成功细胞，挑选8个单细胞克隆并扩增培养，加入强力霉素诱导espl表达。用RT-PCR检测不同浓度强力霉素诱导以及强力霉素诱导不同时间的espl的表达量，并采用传统秋水仙素方法以及流式细胞技术检测诱导不同时间段A549／Ptet-on／TK-Hyg／PTRE-EGFP-espl细胞克隆染色体数目的改变。结果100～200ng／ml浓度范围的强力霉素诱导espl表达量最高，诱导1h即可有espl基因的表达，至48h表达量到达高峰之后逐步减少。在诱导24h后逐步出现染色体数目减少。结论上调espl基因对肿瘤细胞异常染色体有一定的抑制作用，对肿瘤治疗有潜在的应用价值。

Objective To construct a conditional expression vector containing espl in order to observe the effect on the ploidy in A549 cells. Methods Tet-on regulating plasmid was transferred into A549 cells under the Dox induced Tet on regulating system and stable expression of Tet-on was established in An49 cells through G418 selection. The response plasmids of recombinant PTK Hyg and PTRE-EGFP-espl were transferred into positive A549/Tet-on cells,and stable expression of espl was established through hygromycin and green fluorescence selection. Eight single cellular clones were taken and cultivated in amplification. Positive A549/Tet on/TK-Hyg/IRE-EGFP-espl cells,whose expression were induced by Dox showed well dose-response in expression of espl with different concentration and time of Dox induction, were selected by reverse transcriptase-polymerasc chain reaction（RT-PCR）. The changes of chromosome in different A549/Tet-on/TK-Hyg/IRE-EGFP-espl cell clone which were induced different time were detected by the traditional colcbicines method and flow cytometry （FCM）. Results The best Dox concentration induced high cspl expression is from 100ng/ ml to 200ng/ ml. The espl could express after lb induce by Dox and the high expression occured after 48b induction,after that the expression of espl begin to descent. The chromosome of positive A549/Tet-on/TK-Hyg/IRE-EGFP-espl cells had no changes until were induced 24h by Dox. Conclusion The up-regulated expression of espl has certain inhibitory effect on abnormal division of tumor chromosome,so it has potential value in the treatment of cancer.