溶瘤腺病毒DD3-ZD55-SPAG9有效抑制前列腺癌LNCaP细胞移植瘤的生长

Oncolytic adenovirus DD3-ZD55-SPAG9 can effectively inhibit the growth of prostate cancer LNCaP cell xenograft tumor

目的观察差异显示编码3(DD3)启动子调控的携带精子相关抗原9(SPAG9)基因短发夹RNA(shRNA)的新型溶瘤腺病毒(DD3-ZD55-SPAG9)对激素依赖性前列腺癌LNcap细胞系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,当肿瘤体积为100 mm3随机分成3组,空白对照组(PBS组)、对照病毒组(ZD55-SPAG9组)、病毒组(DD3-ZD55-SPAG9组)。测量肿瘤体积,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。原位缺口末端标记法(TUNEL)检测肿瘤组织内细胞凋亡。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和Caspase-8蛋白的表达。采用方差分析(One way-ANOVA),组间比较采用SNK法。结果成功构建裸鼠LNcap细胞移植瘤模型。DD3-ZD55-SPAG9组、ZD55-SPAG9组和PBS组移植瘤终体积分别为(1104.04±110.39)、(1003.20±150.89)和(2321.36±173.87)mm^(3)。DD3-ZD55-SPAG9组和ZD55-SPAG9组肿瘤体积均小于PBS组,差异有统计学意义(F=203.997,P<0.05),但DD3-ZD55-SPAG9组、ZD55-SPAG9组之间差异无统计学意义(P>0.05)。HE染色结果显示,DD3-ZD55-SPAG9组和ZD55-SPAG9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,两者差异无统计学意义(P>0.05)。TUNEL结果显示,DD3-ZD55-SPAG9组和ZD55-SPAG9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,凋亡信号强度明显高于PBS组,差异有统计学意义(F=80.932,P<0.05),两者之间差异无统计学意义(P>0.05)。免疫组织化学法检测肿瘤组织中Caspase-3和Caspase-8蛋白表达的结果显示,DD3-ZD55-SPAG9组和ZD55-SPAG9组与PBS组比较,Caspase-3和Caspase-8蛋白表达量增多,差异有统计学意义(F=73.848、61.514,P<0.05),两者之间差异无统计学意义(P>0.05)。结论DD3-ZD55-SPAG9可以有效抑制前列腺癌LNCaP细胞皮下移植瘤的生长,与对照病毒ZD55-SPAG9比较差异无统计学意义,其机制包括抑制肿瘤细胞生长、诱导凋亡。

Objective To observe the therapeutic effect of a novel oncolytic adenovirus DD3-ZD55-SPAG9 on xenograft of hormone dependent prostate cancer LNCaP cell line in nude mice.Methods The subcutaneous xenograft tumor model of LNCaP cells in nude mice was constructed.When the tumor volume was 100 mm3,the animals were randomly divided into 3 groups:blank control group(PBS group),control virus group(ZD55-SPAG9 group)and virus group(DD3-ZD55-SPAG9 group).Tumor volume was measured and tumor time-volume growth curve was plotted.Hematoxylin and eosin(HE)staining was used to observe the cell growth of xenografts.Terminal-deoxynucleotidyl transferase mediated nick end labeling(TUNEL)analysis was used to detected the cell apoptosis of each group.Immunohistochemistry was done to detect the expression of apoptosis-related proteins including cysteinyl aspartate-specific protease(Caspase)-3 and Caspase-8.One way-ANOVA and SNK method were used for comparison between groups.Results The xenograft tumor model of nude mouse was successfully constructed.The final volume of xenograft tumor in DD3-ZD55-SPAG9 group,ZD55-SPAG9 and PBS group was(1104.04±110.39),(1003.20±150.89) and (2321.36±173.87)mm^(3),respectivenly.Tumor volume in both the DD3-ZD55-SPAG9 group and the ZD55-SPAG9 group was smaller than that in the PBS group,and the difference was statistically significant(F=203.997,P<0.05),and there was no significant significance between DD3-ZD55-SPAG9 group and ZD55-SPAG9 group.HE staining revealed there were a mass of tumor cells scattered in nests or clusters with the atypical and enlarged cell nuclei containing prominent nucleoli in both DD3-ZD55-SPAG9 group and ZD55-SPAG9 group,and there was no significant difference between the two groups.TUNEL results showed that both the DD3-ZD55-SPAG9 group and the ZD55-SPAG9 group showed more red signals under fluorescence in tumor tissue sections,suggesting cell apoptosis,and the intensity of apoptosis signal was significantly higher than that in the PBS group(F=80.932,P<0.05),and there was no significant difference between the two groups.Immunohistochemical staining showed that the expression levels of Caspase-3 and Caspase-8 were significantly up-regulated in both DD3-ZD55-SPAG9 and ZD55-SPAG9 as compared with those in the PBS group(F=73.848,61.514,P<0.05)and there was no significant difference between DD3-ZD55-SPAG9 and ZD55-SPAG9 groups.Conclusion DD3-ZD55-SPAG9 can effectively inhibit the growth of xenograft tumor in prostate cancer LNCaP cells,and has no significant difference compared with the control virus ZD55-SPAG9.The possible mechanisms include inhibition of tumor cell growth and induction of apoptosis.The addition of DD3 promoter did not affect the antitumor activity of oncolytic adenovirus.