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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 investigate retrovirus-mediated endostatin gene transfer and its expression product human endostatin on human vascular endothelial cells. Methods The retroviral vector was used to transduce endostatin gene into lung adenocarcinoma A549 cells. A549 / Endo cells containing endostatin gene were obtained. PCR method to examine the integration of the exogenous endostatin gene in the host cell. A549 / Endo cells were cultured and supernatants collected. The concentration of endostatin in the supernatant was detected by ELISA. MTT, FCM and TUNEL were used to detect the effect of the supernatant on the expression of EA hy926 Inhibition. Results PCR confirmed the integration of exogenous endostatin gene in A549 / Endo cell genome. After cultured for 48h in A549 / Endo cells (1 × 105 / ml), the content of endostatin in the supernatant was (348 ± 120) pg / ml. MTT assay showed that endostatin-containing supernatant could significantly inhibit EA hy926 cells Growth. Endostatin can induce EA hy926 apoptosis by FCM and TUNEL. Conclusion The retrovirus can efficiently mediate endostatin gene transfer. The product of endostatin gene expression can significantly inhibit the growth of human vascular endothelial cells and induce their apoptosis.