以超声微泡为载体的胞嘧啶脱氨酶：尿嘧啶磷酸核糖转移酶融合自杀基因在食管癌细胞株EC9706中的表达

Ultrasound microbubble-mediated cytosine deaminsae： uracil phosphoribosyltransferase suicide gene for esophageal cancer in vitro

目的 观察超声微泡（UM）介导的胞嘧啶脱氨酶：尿嘧啶磷酸核糖转移酶/5-氟胞嘧啶（CD：UPRT/5-FC）自杀基因系统对人食管癌细胞株EC9706的转染效率和对细胞活力的影响.方法 将EC9706细胞按1×108/L接种于12孔孔板中,随机分为4组：A组：空白对照组;B组：单纯质粒组;C组：单纯超声微泡组;D组：超声微泡＋质粒组.24 h后在荧光显微镜下观察各组细胞绿色荧光蛋白的表达,流式细胞术（FCM）检测各组的转染效率,Western blot法检测各组细胞CD基因的表达,细胞计数试剂盒（CCK-8）法检测5-氟胞嘧啶（5-FC）对转染后细胞的杀伤作用.结果 （1）D组细胞中的绿色荧光强度明显高于其他各组,FCM检测各组转染成功率分别为0％、1.42％、0％、32.60％,超声微泡介导的转染对EC9706细胞活性无明显影响（P＞0.05）;（2）Western blot结果显示D组细胞CD基因蛋白条带灰度值明显高于其他各组（P＜0.05）;（3）5-FC对转染后的肿瘤细胞有明显的杀伤作用,细胞存活率明显下降.结论 采用超声微泡携带CD：UPRT融合自杀基因可显著提高在食管癌细胞中的转染效率,增强5-FC对肿瘤细胞的杀伤作用.

Objective To explore the transfection efficiency of cytosine deaminsae：uracil phosphoribosyltransferase/5-flurocytosine （CD：UPRT/5-FC） suicide gene system mediated by ultrasound microbubble （UM） for esophageal cancer in vitro,and indentify the effects on the cell viability.Methods EC9706 cells（l × 108/L） were seeded in the 12-well plates,and randomly divided into 4 groups：A.blank control group; B.CD：UPRT gene only; C.UM only; D.CD：UPRT gene and UM.After transfection,the expression of green fluorescent protein （gfp） was observed under the fluorescent microscopy and quantified by flow cytometry.Western blotting was used to detect the expression of CD protein.Different concentrations （0-160 mg/L） of 5-FC were added into the culture medium,then cells were cultured for another 36 h.The viability of EC9706 cells was measured by CCK-8 assay.Results （1） Gene fluorescence intensity and tansfection efficiency in group D were higher than other groups.The gene transfection efficiency in groups A,B,C,D and E was 0%,1.42%,0% and 32.60% respectively.The cell viability had no significant difference among groups before addition of 5-FC ; （2） CD protein in group D was higher than other groups （P 〈 0.05）.（3） 5-FC had strong antitumor effect on successfully transfected esophageal cancer cells,almost all EC9706 cells in group D were killed when the concentration of 5-FC was above 80 mg/L.Conclusion Ultrasound microbubble can enhance the efficiency of gene transfection without obvious damage to cell viability in EC9706 cells.The method can also enhance the killing effect of CD：UPRT/5-FC suicide gene system in vitro.