视黄酸依赖Fas/RARa融合基因转导靶细胞启动的细胞自杀效应研究

通过构建含Fas抗原“死亡区”和视黄酸核受体RAR配体结合区的融合基因表达载体，使该基因表达受控于视黄酸反应元件（RARE）相连的TK启动子，将Fas/RARa融合基因转导入耐药早幼粒白血病细胞内，进而用视黄酸启动配体依赖细胞自杀机制，清除诱导分化治疗耐药的抗性细胞。该项目在于采用一全新的基因治疗策略有效解决肿瘤诱导分化治疗中的耐药问题。

In this paper, we constructed a novel expression vector of fusion gene Fas/RAR alpha containing RARE-TK promoter and reporter gene eGFP controlled by ATRA and observed the effect of apoptosis initiated by fusion protein Fas/RAR alpha after transfection to HL-60 and MCF-7 malignant cells. The results and conclusions are summarized as follows: 1) The reporter gene plasmid containing RARE (pRARE3-TK-eGFP) was transfected into HL-60 cells. After treatment with ATRA, the expression of eGFP was detected by fluorescence microscopy and RT-PCR. Treatment of transfected HL-60 cells with RA for scheduled times resulted in obvious increase of eGFP in RA dependent manner. These findings suggested that RA is able to induce the expression of exogenous gene under RARE-TK promoter. 2) The fusion gene vector (pRARE-Fas/RAR alpha) containing RARE-TK promoter and eGFP was constructed and transfected to HL-60 and MCF-7 cells. Transfected cells were treated with RA for 24h, 48h, and 72h respectively, as compared with nontransfected ones treated with RA at the same time. The expression of reporter gene (fusion gene) in pRARE-Fas/RAR alpha transfected cells stimulated with RA was higher than that in control group. And it was found that the fusion protein Fas/RAR alpha was mainly located in cell membrane. 3) The proliferation capacity was decreased, the apoptotic rate and the DNA fragmentation rate were increased in cells treated by RA. The DNA ladder was detectable in RA-treated cells. The morphological apoptosis changes were observed by transmission electron microscopy and TUNEL detection. These changes were more obviously in pRARE-Fas/RAR alpha transfected cells than nontransfected cells. Our study demonstrated that RA and the product of exogenous fusion gene induced by RA could cooperate in inhibition of cell growth and inducing apoptosis.4) The expressions of caspase-8 and caspase-3 genes were increased in tumor cells treated with RA. The expression of active subunit protein of caspase-3 was increased in HL-60 cells treated with RA. These changes were more obvious in transfected cells than nontransfected cells. Our study suggested that up-regulated expression and activity of several caspases may be play a key role in RA and fusion gene inducing apoptosis..The results above are helpful to find the optimal strategies to overcome the resistance to RA and decrease ill effects in clinical use of RA. .