靶向Her2/neu基因小干扰RNA对肺腺癌Calu-3细胞株药物敏感性的影响  

The effect of siRNA-Her2/neu on the drug sensitivity of Her2/neu-overexpressing lung adenocarcinoma cell line

作　　者：任淑华[1] 张林[1] 王井伟[2] 田海梅[3] 曲平[3] 张伟[3] 

机构地区：[1]中国医科大学第一附属医院胸外科,沈阳110001 [2]唐山工人医院外科 [3]中国医学科学院中国协和医科大学肿瘤研究所肿瘤生物学检测中心

出　　处：《中华结核和呼吸杂志》2006年第1期35-38,共4页Chinese Journal of Tuberculosis and Respiratory Diseases

基　　金：国家自然科学基金资助项目(30371624)

摘　　要：目的探讨转染靶向Her2/neu基因小干扰RNA(sm all interfering RNA,siRNA)对肺腺癌Calu-3细胞株药物敏感性的影响。方法实验分4组,未转染对照组、空载体组、非特异性siRNA组、Her2/neu siRNA组,实验重复5次。即用Her2/neu基因的特异性siRNA和非特异性siRNA通过阳离子脂质体L ipofectAM INE 2000分别转染Calu-3细胞。采用流式细胞仪(FCM)检测各组Calu-3细胞Her2/neu蛋白和P糖蛋白(P-gp)的表达水平,四甲基偶氮唑蓝(MTT)法检测顺铂(DDP)对转染siRNA的癌细胞增殖水平的影响,应用膜联蛋白Ⅴ-异硫氰酸荧光素(AnnexinⅤ-FITC)凋亡试剂盒检测各组Calu-3细胞的凋亡水平。结果Her2/neu siRNA组Calu-3细胞Her2/neu蛋白表达的阳性率为(25.04±1.56)%、P-gp蛋白表达阳性率为(4.24±1.01)%,而未转染对照组、空载体组、非特异性siRNA组Her2/neu蛋白表达的阳性率分别为(98.24±2.23)%、(95.67±1.98)%、(94.79±0.87)%;P-gp蛋白表达阳性率分别为(5.11±2.98)%、(6.98±2.47)%、(5.59±3.66)%。Her2/neu siRNA组Calu-3细胞Her2/neu蛋白表达受到明显抑制(F=112,P<0.05);而P-gp的表达水平各组差异无统计学意义(F=2.45,P>0.05);Her2/neu siRNA联合DDP作用的细胞抑制率达(67.1±2.3)%,而未转染对照组、空载体组及非特异性siRNA组联合DDP细胞抑制率分别为(48.1±3.5)%、(46.3±5.9)%及(50.2±2.9)%,3组间抑制率差异无统计学意义(F=8.68,P>0.05),3组分别与Her2/neu siRNA组比较差异有统计学意义(P<0.01);FCM分析显示靶向Her2/neu基因特异性siRNA组Calu-3细胞的DDP诱导凋亡率为(35.6±3.4)%,明显高于未转染对照组的(8.8±0.5)%、空载体组的(9.6±1.7)%及非特异性siRNA组的(11.3±1.8)%,差异有统计学意义(F=10.68,P<0.01)。结论靶向Her2/neu基因siRNA能增强肺腺癌Calu-3细胞株对顺铂的敏感性。Objective To explore the effect of synthesized siRNA targeting Her2/neu oncogene on the drug sensitivity of Her2/neu-overexpressing lung adenoeareinoma cell line. Methods The experiments consisted of four groups including an untreated control group, an empty vector group , an unrelated siRNA group, and a Her2/neu siRNA group. Every experiment was repeated five times. Lung adenoeareinoma cell line Calu-3 was transfected with siRNAs formulated LipofeetAMINE 2000, and Her2/neu protein and P-gp of each group were determined by flow eytometry （FCM）. The ehemosensitivity of transfeeted cells to eisplatin （DDP） was measured by MTT. Cell apoptosis detection kit （AnnexinⅤ method） was used to examine the drug induced apoptosis rate. Results The Her2/neu protein and P-gp positive expression rate in the Her2/ neu siRNA group, the untreated control group, the empty vector group and the unrelated siRNA group were [（25.04±1.56）%,（4.24±1.01）%],[（98.24±2.23）%,（5.11 ±2.98）%],[（95.67 ±1.98）%, （ 6.98 ± 2.47 ） % ] or [ （ 94. 79 ± 0. 87 ） % , （ 5.59 ± 3.66 ） % ], respectively. Introduction of the sequence specific siRNA into Her2/neu positive Calu-3 cells in vitro greatly reduced the cell surface expression of the Her2/neu protein, but had no effect on P-gp level. Consequently the inhibitory rate of DDP in combination with siRNA targeting Her2/neu was （ 67. 1 ± 2. 3 ） % , but it was （48. 1 ± 3.5 ） % , （46. 3 ± 5.9 ） % and （50. 2 ±2.9）% in the untreated control, the empty vector and the unrelated siRNA groups, respectively. There was a significant difference between Her2/neu siRNA group and other three groups （P 〈 0. 01 ）. The FCM results showed the apoptosis rate of DDP combined with siRNA-Her2/neu was elevated as compared with the unrelated siRNA group, the empty vector group or the untreated control group. Conclusion Sequence specific siRNA-Her2/neu was capable of enhancing the chemosensitivity of Calu-3 cells to cisolatin.

关 键 词：肺肿瘤 基因 ERBB-2 顺铂 小干扰RNA 

分 类 号：R734.2[医药卫生—肿瘤]