摘要目的 探讨尾型同源盒基因2(Cdx2)沉默对逆转人胃癌耐顺铂细胞SGC7901/DDP多药耐药性的影响.方法 将对数生长期的人胃癌耐顺铂细胞SGC7901/DDP接种于培养板中,分为3组:感染RNA干扰Cdx2基因沉默重组慢病毒载体(pLL-Cdx2-shRNA)作为实验组；感染慢病毒空载体作为阴性对照组；空白对照组不做任何处理.Western blot及RT-PCR检测Cdx2及凋亡相关基因c-myc、cyclin D1、survivin等蛋白及mRNA表达水平；MTT法检测3组细胞对化疗药物阿霉素、5-氟尿嘧啶、顺铂的敏感性；流式细胞技术检测3组细胞阿霉素的泵出率、细胞周期分布及细胞凋亡情况.计量资料用x±s表示,多组间比较采用单因素方差分析,两组间比较采用SNK-q检验,计数资料采用x2检验.结果 实验组、阴性对照组、空白对照组人胃癌耐顺铂细胞SGC7901/DDP中各蛋白相对表达量:Cdx2分别为0.187 ±0.060、0.535±0.033、0.567±0.014,c-myc分别为0.086±0.004、0.379±0.006、0.354±0.004,cyclin D1分别为0.016±0.005、0.141±0.003、0.162 ±0.008,survivin分别为0.276±0.012、0.672±0.009、0.517±0.313,与阴性对照组和空白对照组比较,实验组上述蛋白表达量明显下降,差异有统计学意义(F =247.385,3.353,597.882,98.628,P＜0.05).实验组、阴性对照组、空白对照组mRNA相对表达量:Cdx2分别为0.184±0.010、0.894±0.056、0.837±0.049,c-myc分别为0.212±0.022、0.538±0.021、0.545±0.032,cyclin D1分别为0.045±0.009、0.163±0.009、0.157 ±0.010,survivin分别为0.401±0.027、0.824±0.016、0.782±0.056,与阴性对照组和空白对照组比较,实验组上述基因mRNA表达量明显下降,差异有统计学意义(F=243.776,161.793,138.523,118.426,P＜0.05).MTT法检测实验组、阴性对照组和空白对照组人胃癌耐顺铂细胞SGC7901/DDP阿霉素IC50值分别为(0.12±0.05)mg/L、(0.33±0.08)mg/L、(0.39±0.15) mg/L,5-氟尿嘧啶IC50值分别为(0.52±0.13) mg/L、(4.10±1.25) mg/L、(4.05±1.44) mg/L,顺铂IC50值分别为(0.82±0.13) mg/L、(2.81 ±0.50) mg/L、(3.28±1.03) mg/L.实验组、阴性对照组、空白对照组中人胃癌耐顺铂细胞SGC7901/DDP对阿霉素泵出率分别为0.21％、0.37％和0.35％.与阴性对照组和空白对照组比较,实验组人胃癌耐顺铂细胞SGC7901/DDP对阿霉素、5-氟尿嘧啶和顺铂的IC50值及对阿霉素的泵出率显著降低,差异均有统计学意义(F=8.101,13.854,15.159,x2=7.106,P＜0.05).实验组、阴性对照组与空白对照组人胃癌耐顺铂细胞SGC7901/DDP G0/G1期细胞比例分别为17.87％、34.71％、37.20％,与阴性对照组和空白对照组比较,实验组G0/G1期细胞减少,差异有统计学意义(x2=1.055,P＜0.05)；实验组G2/M期的细胞比例为11.93％,细胞凋亡率为31.13％,均明显高于阴性对照组的0.26％、16.58％和空白对照组的0.35％、13.18％,差异有统计学意义(x2=2.249,11.030,P＜0.05).结论 Cdx2基因沉默可有效提高人胃癌耐顺铂细胞SGC7901/DDP对化疗药物的敏感性,增加化疗药物在胃癌细胞内的蓄积浓度,逆转人胃癌耐顺铂细胞SGC7901/DDP的耐药性.

abstractsObjective To investigate the effects of caudal type homeobox 2 (Cdx2) silence on reservion of multi-drug resistance of gastric carcinoma cisplantin-resistant cell SGC7901/DDP.Methods Gastric carcinoma cisplantin-resistant cells SCG7901/DDP in the logarithmic phase were cultured in the plate,and were divided into the experimental group [gastric carcinoma cells of SGC7901/DDP were infected with a silent Cdx2-recombinanted lentiviral vector (pLL-Cdx2-shRNA)],the negative control group (gastric carcinoma cells of SGC7901/DDP were infected with empty lentiviral vector) and the blank control group (gastric carcinoma cells of SGC7901/DDP were not treated).The protein and mRNA expressions of Cdx2 and apoptosis related genes like c-myc,cyclin D1 and survivin were detected by the Western blot and reverse-transcription PCR,respectively.The sensitivity of the cells in the 3 groups to adriamycin,5-fluorouracil and cisplatium were assessed by MTT.The pump-out rate of adriamycin,cell cycle distribution and apoptosis of the 3 groups were analyzed using flow cytometry.All measurement data were expressed with mean ± standard deviation.Comparison among multi-groups was done by one-way analysis of variance,and comparison between 2 groups was done by SNK-q test.The enumeration data were analyzed using the chi-square test.Results The relative protein expression levels of Cdx2,c-myc,cyclin D1 and survivin were 0.187 ± 0.060,0.086 ± 0.004,0.016 ± 0.005 and 0.276 ± 0.012 in the experimental group,0.535 ± 0.033,0.379 ± 0.006,0.141 ± 0.003 and 0.672 ± 0.009 in the negative control group,and 0.567 ± 0.014,0.354 ± 0.004,0.162 ± 0.008 and 0.517 ± 0.313 in the blank control group,respectively.The relative protein expression levels of Cdx2,c-myc,cyclin D1 and survivin in the experimental group were significantly lower than those in the negative control group and the blank control group (F =247.385,3.353,597.882,98.628,P ＜0.05).The relative mRNA expression levels of Cdx2,c-myc,cyclin D1 and survivin were 0.184 ± 0.010,0.212 ± 0.022,0.045 ± 0.009 and 0.401 ± 0.027 in the experimental group,0.894 ± 0.056,0.538 ± 0.021,0.163 ±0.009 and 0.824 ± 0.016 in the negative control group,and 0.837 ±0.049,0.545 ±0.032,0.157 ±0.010 and 0.782 ±0.056 in the blank control group,respectively.The relative mRNA expression levels of Cdx2,c-myc,cyclin D1 and survivin in the experimental group were significantly lower than those in the negative control group and the blank control group (F =243.776,161.793,138.523,118.426,P ＜ 0.05).The IC50 values detected by MTT of adriamycin,5-flurouracile and cisplatin to gastroc carcinoma cisplantin-resistant cell SCG7901/DDP were (0.12 ± 0.05) mg/L,(0.52 ± 0.13) mg/L and (0.82 ± 0.13) mg/L in the experimental group,(0.33 ± 0.08) mg/L,(4.10.± 1.25) mg/L and (2.81 ± 0.50) mg/L in the negative control group,(0.39 ±0.15)mg/L,(4.05 ± 1.44) mg/L and (3.28 ± 1.03) rng/L in the blank control group,respectively.The pump-out rates of adriamycin of the experimental group,negative control group,and the blank control group were0.21％,0.37％ and 0.35％.Compared with the negative control group and the blank control group,the IC50values of adriamycin,5-fluorouracil and cisplatin in the experimental group were significantly increased,and thepump-out rate of adriamycin was significantly decreased (F =8.101,13.854,15.159,x2 =7.106,P ＜ 0.05).The ratios of cells in the G0/G1 phase were 17.87％,34.71％ and 37.20％ in the experimental group,negative control group and the blank control group,respectively.Compared with the negative control group and blank control group,the ratio of cells in the G0/Gt was significantly decreased (x2=1.055,P ＜ 0.05).The ratio of cells in the G2/M phase in the experimental group was 11.93％,and the apoptosis rate was 31.13％,which were significantly higher than the negative group (0.26％,16.58％) and the blank control group (0.35％,13.18％) (x2=2.249,11.030,P ＜ 0.05).Conclusions Silent Cdx2 can effectively enhance the sensitivity of the SGC7901/DDP cells and the intracellular accumulation concentration of the drugs.Silent Cdx2 can also reverse the multidrug resistance of the SGC7901/DDP cells.