Survivin反义核酸协同紫杉醇的体外及大鼠体内抗肿瘤作用

In Vitro and in Vivo Synergetic Antitumor Effect of Survivin Antisense Oligodeoxynucleotides and Taxol

目的研究转染生存蛋白survivin反义寡核苷酸的抗肿瘤作用,对大鼠胃癌模型形成的干预作用、机制及是否与紫杉醇间有协同作用。方法在脂质体介导下进行survivin反义寡核苷酸的体外转染,以WesternBlot检测survivin蛋白表达,以MTT法检测转染survivin反义寡核苷酸及联合紫杉醇对胃癌BGC823细胞生长增殖的影响;以高盐饲料和甲基硝基二硝基胍(MNNG)诱导大鼠发生胃癌,造模中期分别腹腔注射脂质体包裹的survivin反义寡核苷酸、紫杉醇或两者联用,检测造模结束时各组大鼠胃癌的发生率、肿瘤组织中Brdu标记率及凋亡指数。结果Survivin反义寡核苷酸能明显抑制survivin蛋白表达,并抑制BGC823细胞的生长,其IC50约350nmol/L;survivin反义寡核苷酸与紫杉醇联合能显著降低其IC50值,起到协同抗肿瘤作用。造模结束时对照组(C)、紫杉醇组(T)、反义寡核苷酸组(A)、反义寡核苷酸联合紫杉醇组(AT)胃腺癌发生率分别为90%、78.6%、73.3%和50%,AT组的胃癌发生率显著低于对照组(P<0.05),两个单独治疗组胃癌发生率均低于对照组,但无统计学差异(P>0.05);AT组胃癌的BrdU标记率(10.2±4.7)显著低于对照组(18.7±5.5,P<0.05)及T、A两个单独治疗组(分别为16.0±5.8和15.4±5.2,P<0.05);AT组胃癌的凋亡率(1.71±0.41)显著高于对照组(0.91±0.14,P<0.01)及T、A单独治疗组(1.19±0.42、1.29±0.43,P<0.05)。结论Survivin反义寡核苷酸能抑制BGC823细胞生长,与紫杉醇有协同抗肿瘤作用;腹腔注射survivin反义寡核苷酸与紫杉醇能协同抑制胃癌形成,与促进凋亡和抑制细胞增殖有关。Survivin是抗肿瘤基因治疗的良好靶点。

Purpose To identify the antitumor effect of survivin antisense oligodeoxynueleotides （AS-ODN） in vitro and in vivo .and whether synergism exists between survivin AS-ODN and taxol. Methods Sense and antisense survivin oligodeoxynueleotides were transfeeted into BGC-823 cells by lipofeet amine mediated transient transfeetion method. Taxol was administrated to both transfeeted and untransfeeted cells. Cell proliferation was measured by MTT method and survivin expression was detected by Western Blot. In animal experiment ,65 six-week-old Wistar rats were fed with N-methyl-N＇-nitro-N-nitrosoguanidine （MNNG） solutions and a diet supplemented with 8 % NaCl in order to induce gastric cancer. At the end of week 20, these rats were divided into 4 groups randomly： the control group （C）,20 rats, no therapy were used; the taxol group（T）, 15 rats were given ip injections of taxol at a dose of 10 mg/kg every other week; the antisense group（A）. 15 rats were given ip injections of an survivin AS-ODN lipofeet amine complex at a dose of 50 μg/kg every 72 hours;taxol associated with antisense group（AT）, 15 rats were given i.p. injections of taxol and survivin AS-ODN as group T and A. At the end of week 35,all the surviving rats were killed. The incidence of gastric cancer in each group were calculated. The Brdu labeling index and apoptosis index in gastric cancer tissues of each group were detected with immunohistochemical analysis and TUNEL, respectively. Results About 50 % reduction of survivin expression and inhibition of cell prolife-ration with an IC50 at 350 nmol/L were noted in the survivin AS-ODN transfected cells while little effects noted in the sense ODN transfected cells. The combination of transfection survivin AS-ODN with taxol significantly reduced the IC50 of taxol from 60 ng/mL to 2.4 ng/mL. The incidence of gastric cancer in C, T, A, and AT group is 90 % , 78.6 % ,73.3 % , and 50 % respectively. The incidence of AT group was significantly lower than that of C group（ P 〈0.05）. The BrdU labeling index of AT was 10.2± 4.7, and was significantly lower than that of C group（18.7 ± 5.5,P〈0.05） and T,A group （16.0± 5.8,15.4 ± 5.2,P〈0.05）. The apoptosis index of gastric cancer tissues in AT group were 1.71 ± 0.41 ,and were significantly lower than that of C group （0.91±0.14） andT,A group （1.19±0.42,1.29±0.43,P〈0.05）. Conclusions Both in vitro and in vivo,combination of taxol with transfection survivin AS-ODN can get synergetic antitumor effect by reducing cell proliferation and augmenting apoptosis. This suggests that survivin is an effective target of antitumor therapy.