内皮抑素基因表达产物对血管内皮细胞生长的抑制作用

The suppressive effects of the expressed product of h-endostatin gene on the proliferation of vascular endothelial cell

目的探讨逆转录病毒介导内皮抑素基因转移的方法及其表达产物人内皮抑素对人血管内皮细胞的作用。方法以逆转录病毒为载体,转导内皮抑素基因至肺腺癌A549细胞,获得含内皮抑素基因的A549/Endo细胞。PCR方法检验外源性内皮抑素基因在宿主细胞中的整合。培养A549/Endo细胞,收集上清液。ELISA法检测上清液中内皮抑素的浓度,四唑盐比色试验(MTT)、流式细胞术(FCM)、原位细胞凋亡(TUNEL)观察该上清液对血管内皮细胞EA hy926 的抑制作用。结果PCR法证实A549/Endo细胞基因组中整合有外源性内皮抑素基因。A549/Endo细胞(1×105/ml)培养48h后,上清液中内皮抑素含量为(348±120) pg/ml,MTT法显示含内皮抑素的上清液能明显抑制EA hy926细胞的生长。FCM、TUNEL法显示内皮抑素可诱导EA hy926 凋亡。结论逆转录病毒能高效介导内皮抑素基因转移,内皮抑素基因表达产物能明显抑制人血管内皮细胞的生长,诱导其凋亡。

Objective To establish a method of retrovirus-mediated h-enodstatin gene transfer and to estimate the suppressive effects of its expressed product on the proliferation of vascular endothelial cell. Methods H-endostatin gene was transferred into lung adenocarcinoma A549 cell by the vector of retrovirus to obtain a A549/Endo cell . PCR was used to confirm the transfer of endostatin gene. Its expressed product in the supernatant of A549/Endo cells, the endostatin, was detected by ELISA.And the suppressive effects of h-endostain on the proliferation of endothelial cell EA.hy926 was estimated by the methods of MTT,flow cytometry (FCM) , and TUNEL stains.Results PCR proved that the h-endostatin gene was successfully inserted into A549 genomic DNA. ELISA showed that the h-endostain in the supernatant of culture medium of A549/Endo cells (1×105/ml) was about 348±120pg/ml at 48 hour after the cells were cultivated. MTT confirmed that the h-endostatin in the supernatant of A549/Endo cells could effectively inhibit the proliferation of endothelial cell EA.hy926. Moreover, the FCM and TUNEL further confirmed that the h-endostain could induce the apoptosis of EA.hy926 cell.Conclusion The endostatin gene can be highly transferred into A549 cells by retroviruses . H-endostatin can significantly inhibit the vascular endothelial cell proliferation and induce the cell apoptosis.