溶瘤腺病毒荷载核基质结合区结合蛋白1短发卡RNA联合多西他赛治疗前列腺癌LNcap细胞移植瘤
Inhibitory effects of oncolytic virus carrying short hairpin RNA targeting special AT rich sequence binding protein-1 combined with docetaxel on hormone-sensitive prostate cancer LNCaP xenografts
目的:观察荷载核基质结合区结合蛋白1(SATB1)短发卡RNA(shRNA)溶瘤腺病毒联合多西他赛(DTX)对激素依赖性前列腺癌LNCaP细胞株裸鼠移植瘤的治疗作用。方法:构建裸鼠前列腺癌LNCaP细胞皮下移植瘤模型(裸鼠购自上海西普尔-必凯实验动物有限公司),随机分成4组:空白对照组(PBS组)、化疗组(DTX组,20 mg/kg)、病毒治疗组(ZD55-SATB1组,1×10 8 pfu)、病毒联合化疗组(ZD55-SATB1+DTX组,5×10 7 pfu+10 mg/kg)。绘制肿瘤时间-体积生长曲线。苏木精-伊红(HE)染色观察肿瘤细胞的生长。免疫组织化学检测肿瘤组织中半胱氨酰天冬氨酸特异性蛋白酶(Caspase)-3、Caspase-8、B细胞淋巴瘤/白血病-2(bcl-2)和CD31蛋白的表达。脱氧核糖核苷酸末端转移酶介导的缺口末端标记法(TUNEL)检测肿瘤组织细胞凋亡。采用方差分析(One way-ANOVA),组间比较采用 t检验。 结果:成功构建裸鼠LNCaP细胞移植瘤模型。病毒联合化疗组、病毒治疗组、化疗组和空白对照组移植瘤终体积分别为(616.23±101.56)、(938.59±102.98) mm 3( t=3.860, P<0.05)、(1 206.92±202.17) mm 3( t=4.522, P<0.05)和(2 254.05±188.99) mm 3( t=13.220, P<0.01),差异均有统计学意义。HE染色结果显示,病毒联合化疗组、病毒治疗组和化疗组的肿瘤组织中均可见较多的死细胞,细胞裂解成碎片,细胞核固缩碎裂,其正常的组织结构消失,但联合组的肿瘤细胞坏死更明显。免疫组织化学法结果显示化疗组、病毒治疗组、病毒联合化疗组内Caspase-8、Caspase-3、bcl-2和CD31的相对蛋白表达量分别为2.11±0.41、1.67±0.21、0.65±0.06、0.78±0.05( t=4.136、10.930、5.523、8.162, P<0.05);2.39±0.33、2.61±0.51、0.56±0.06、0.68±0.04( t=5.594、4.119、3.242、6.010, P<0.05);3.42±0.37、3.61±0.23、0.44±0.02、0.50±0.04,联合组与单用药物或病毒组比较,Caspase-8、Caspase-3蛋白表达量增多,bcl-2蛋白表达量降低,差异有统计学意义。联合组移植瘤组织中CD31蛋白的表达量与单用药物或病毒组比较显著降低,说明联合组能有效促进促凋亡蛋白的表达,降低抑凋亡蛋白的表达,且在一定程度上抑制肿瘤组织血管的生成。TUNEL结果显示:化疗组、病毒治疗组、病毒联合化疗组的肿瘤组织切片荧光下均可见较多红色信号,提示细胞凋亡,各组凋亡率分别为(26.17±2.97)%( t=9.557, P<0.01)、(35.68±3.46)%( t=3.931, P<0.05)、(44.07±1.30)%,联合组内凋亡细胞更为明显,差异有统计学意义。 结论:溶瘤腺病毒和多西他赛均可抑制前列腺癌LNCaP细胞皮下移植瘤的生长,且联合效果优于单一用药,其可能的机制包括抑制肿瘤细胞生长、诱导凋亡、抑制血管生成。
更多Objective:To investigate the curative effectiveness of oncolytic adenovirus carrying short hairpin RNA (shRNA) targeting special AT rich sequence binding protein-1 (SATB1) (ZD55-SATB1) combined with docetaxel for hormone-sensitive prostate cancer LNCaP xenografts.Methods:The human prostate cancer LNCaP cells xenograft models in nude mice were established, and divided into four groups: control group (PBS), drug treatment group (DTX group; 20 mg/kg), adenovirus treatment group (ZD55-SATB1 group; 1×10 8 pfu), adenovirus combined with drug treatment group (ZD55-SATB1+ DTX group; 10 mg/kg+ 5×10 7 pfu). The volume of xenografts of nude mice in each group was measured and the tumor time-growth curve was drawn. HE staining was sued to observe the cell growth of xenografts. Immunohistochemistry was performed to detect the expression of apoptosis-related proteins including bcl-2, Caspase-3, Caspase-8 and CD31. TUNEL analysis was done to detect the cell apoptosis of each group. Results:The xenograft tumor models of nude mice were successfully constructed. The average tumor size in the ZD55-SATB1+ DTX group was (616.23±101.56) mm 3, which was significantly smaller than that in the ZD55-SATB1 group [(938.59±102.98) mm 3, t=3.860, P<0.05], the DTX group [(1 206.92±202.17) mm 3, t=4.522, P<0.05], and PBS group [(2 254.05±188.99) mm 3, t=13.220, P<0.01]. The xenograft tumor volume in adenovirus combined with drug treatment group was smaller than in control group and monotherapy group with the differences being statistically significant. HE staining revealed that there were a mass of tumor cells scattered in nests or clusters with the atypical and enlarged cell nuclei containing prominent nucleoli in DTX group, ZD55-SATB1 group, ZD55-SATB1+ DTX group. The number of necrosis tumor cells was more in ZD55-SATB1+ DTX group. Immunohistochemical staining showed that the expression levels of Caspase-8, Caspase-3, bcl-2 and CD31 in DTX group, ZD55-SATB1 group, ZD55-SATB1+ DTX group were 2.11±0.41, 1.67±0.21, 0.65±0.06 and 0.78±0.05 ( t=4.136, 10.930, 5.523, 8.162, P<0.05); 2.39±0.33, 2.61±0.51, 0.56±0.06 and 0.68±0.04 ( t=5.594, 4.119, 3.242, 6.010, P<0.05); 3.42±0.37, 3.61±0.23, 0.44±0.02 and 0.50±0.04, respectively. The expression of Caspase-8/3 protein was up-regulated, and that of bcl-2 protein was down-regulated in combination group as compared with the DTX group or oncolytic adenovirus treatment group alone with the differences being statistically significant. Moreover, the expression of CD31 protein in the combination group was down-regulated, which indicated that the combination group could accelerate the expression of pro-apoptotic proteins, decrease the expression of inhibitor of apoptotic proteins and inhibit the formation of tumor tissue vessels. The TUNEL results demonstrated that the fluorescent staining was seen in combination group, DTX group and oncolytic adenovirus treatment group, and the apoptosis rate in DTX group, oncolytic adenovirus treatment group and combination group was (26.17±2.97)% ( t=9.557, P<0.01), (35.68±3.46)% ( t=3.931, P<0.05) and (44.07±1.30)% respectively. The apoptosis was the most in combination group. Conclusion:Both oncolytic adenovirus and docetaxel could remarkably inhibit the growth of xenografts of LNCaP cells and the anti-tumor effect of combination group was more effective than monotherapy group. The mechanisms probably include inhibiting the growth of tumor cells, inducing cell apoptosis and suppressing angiogenesis.
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