荷载肿瘤坏死因子相关诱导凋亡配体基因的溶瘤腺病毒联合吉西他滨对裸鼠人膀胱癌T24细胞移植瘤的抑制作用

Inhibitory effect of gemcitabine and oncolytic adenovirus carrying tumor necrosis factor-related apoptosis-inducing ligand on implanted human T24 bladder cancer T24 in nude mice

目的 观察荷载肿瘤坏死因子相关诱导凋亡配体基因的溶瘤腺病毒ZD55-肿瘤坏死因子相关诱导凋亡配体（TRAIL）联合吉西他滨（Gemcitabine）对膀胱癌T24细胞移植瘤的抑制作用.方法 T24细胞以2×10^6个（0.2 ml）接种于裸鼠右腋皮下,建立裸鼠膀胱癌T24细胞移植瘤模型,分别给予ZD55-TRAIL[转染倍数（MOI）=10]联合Gemcitabine（4.0g/L）、ZD55-TRAIL（MOI=10）、Gemcitabine（4.0 g/L）、磷酸盐缓冲液（PBS）各10μl连续瘤体内注射3d.每周测量裸鼠肿瘤生长体积,免疫组织化学法检测T24细胞移植瘤组织TRAIL、早期区1A基因（E1A）蛋白表达,原位末端标记法（TUNEL）检测移植瘤细胞凋亡.结果 ZD55-TRAIL联合Gemcitabine能显著抑制肿瘤的生长,干预9周后ZD55-TRAIL联合Gemcitabine、ZD55-TRAIL、Gemcitabine、PBS处理组肿瘤平均体积分别为（129.0±8.3）、（1760.6±83.3）、（1 129.3±73.2）、（2 501.0±221.8） mm3,各组差异有统计学意义（P＜0.01）.免疫组织化学表明,ZD55-TRAIL联合Gemcitabine高效表达TRAIL、E1A蛋白.TUNEL表明：ZD55-TRAIL联合Gemcitabine、ZD55-TRAIL、Gemcitabine、PBS处理组细胞凋亡率分别为（85.8±5.6）％、（61.4±3.8）％、（44.0±3.8）％、（15.8±3.2）％,表明ZD55-TRAIL联合Gemcitabine能有效诱导肿瘤细胞凋亡.结论 ZD55-TRAIL联合Gemcitabine可协同抑制膀胱癌T24细胞移植瘤的生长.

Objective To investigate the inhibitory effect of oncolytic adenovirus carrying tumor necrosis factor-related apoptosis-inducing ligand （TRAIL） and gemcitabine on implanted human T24 cell bladder cancer in nude mice.Methods The bladder cancer xenograft model was established by subcutaneously injecting 2 × 10^6 T24 cells into the right flank of mice.Mice were divided randomly into four groups and treated by intratumoral injection of ZD55-TRAIL [multiple of infection （MOI） =10] plus gemcitabine （4.0 g/L）,ZD55-TRAIL （MOI =10）,gemcitabine （4.0 g/L） which was dissolved in 100 μl saline,or 100 μl phosphate buffer（PBS） as the control,respectively,once every for three consecutive days.The tumor volume was measured every week for 9 weeks.Seven days after the end of the treatment,some mice were sacrificed followed by the determination of TRAIL and early region 1A （E1A） protein levels in tumor tissue by immunohistochemical staining.The apoptosis of tumor xenografts was measured by TdT-mediated dUTP nick end labeling （TUNEL）.Results In the control group,tumors displayed rapid and continued outgrowth during the course of the experiment,with the mean tumor size of （2 501.0 ±221.8） mm3.In sharp contrast,the mean tumor size in the ZD55-TRAIL plus gemcitabine group was （129.0 ± 8.3） mm3,which was significantly smaller than that in the ZD55-TRAIL group [（1 760.6 ± 83.3） mm3,P 〈 0.05] and the gemcitabine group [（1 129.3 ± 73.2） mm3,P 〈 0.05].As compared with the gemcitabine-and PBS-treated groups,there was marked increase of TRAIL staining in the ZD55-TRAIL plus gemcitabine group and ZD55-TRAIL-treated group.Moreover,the E1A expression was detected only in ZD55-TRAIL plus gemcitabine group and ZD55-TRAIL-treated group but not in the gemcitabine group and PBS group.TUNEL staining showed there was significantly increased apoptosis in the ZD55-TRAIL plus gemcitabine group [（85.8 ± 5.6） %] in comparison to that in the PBS-treated group [（15.8 ± 3.2） %],ZD55-TRAIL group [（61.4 ± 3.8） %] or gemcitabine group [（44.0 ± 3.8） %].Conclusion ZD55-TRAIL and gemcitabine have the synergistic anti-tumor effects on bladder cancer in vivo.