双突变溶瘤腺病毒联合吉西他滨治疗裸鼠原位膀胱癌的效果  

Double-mutated oncolytic adenovirus combined with gemcitabine for treating an orthotopic nude mouse model of bladder cancer

作　　者：王华[1] 刘卓[1] 王宗平[1] 李方印[1] 赵阳[1] 陈贵平[1] 李德川[1] 

机构地区：[1]310022杭州,浙江省肿瘤医院泌尿外科

出　　处：《中华肿瘤杂志》2013年第6期412-417,共6页Chinese Journal of Oncology

基　　金：浙江省钱江人才计划项目（2009R10001）

摘　　要：目的探讨双突变溶瘤腺病毒AxdAdB-3联合吉西他滨治疗裸鼠原位膀胱癌的效果。方法以含有LacZ基因的腺病毒AxCAIacZ感染人膀胱癌细胞株YTS-1、T24、5637、KK47和正常细胞株HCV29、W138，用5-溴-4-氯-3-吲哚-β-D-半乳糖苷染色，检测不同细胞株对腺病毒的易感性。以AxCAlacZ和AxdAdB-3感染上述细胞，免疫组化染色检测不同细胞中腺病毒结构蛋白的表达。采用流式细胞术检测经腺病毒感染后YTS-1细胞中S期细胞的比例。采用细胞计数盒8法检测AxdAdB-3和（或）吉西他滨对不同膀胱癌细胞的杀伤能力。建立裸鼠原位膀胱肿瘤模型，观察膀胱内灌注AxdAdB-3和（或）吉西他滨对膀胱肿瘤的生长抑制效果。结果不同的细胞株对腺病毒的易感性不同，5637和KK47细胞株易感性较强，而YTS-1和T24细胞易感性较差。免疫组化染色显示，AxdAdB-3只选择性地在肿瘤细胞内复制，而在正常细胞内不复制。AxCAlacZ或AxdAdB-3感染YTS-1细胞48h后，s期细胞的比例分别为（39±3）％和（49±5）％（P〈0．05）。体外和体内实验的结果均显示，AxdAdB-3和吉西他滨联合使用比单一使用可更加有效抑制膀胱癌细胞的生长，其中AxCAlacZ组、吉西他滨组、AxdAdB-3组和吉西他滨＋AxdAdB-3组的平均瘤重分别为400．6、126．4、82．0和40．4mg（P〈0．001）。结论AxdAdB-3联合吉西他滨膀胱内灌注可有效治疗裸鼠膀胱原位肿瘤，值得进一步开展相关临床研究。Objective To investigate the therapeutic efficacy of double-mutated oncolytic adenovirus AxdAdB-3 in combination with gemcitabine for treating bladder cancer in an orthotopic nude mouse model. Methods The susceptibility to the adenovirus was evaluated in bladder cancer cell lines YTS-1, T24, 5637 and KK47, and normal cell lines HCV29 and WI38. The cells were infected with AxCAlacZ and stained with 5-bromo-4-chloro-3-indolyl-13-galactoside （X-Gal）. Immunostaining against adenoviral hexon protein was performed to determine the selective replication of AxdAdB-3 in the cancer cells. Flow eytometry was used to determine the YTS-1 ceils in S phase of cell cycle after adenovirus infection. Cell viability after AxdAdB-3 and/or gemcitabine was measured by CCK-8 assay. Orthotopie bladder cancer model was established in nude mice, and the inhibitory efficacy of intravesical instillation therapy with AxdAdB-3 or/and gemcitabine was assessed. Results Gene transduction efficiency was different among the cell lines, and correlated with expression of CAR. 5637 and KK47 ceils with high expression of CAR were more susceptible to the adenovirus, whereas YTS-1 and T24 cells with little CAR expression were resistant to adenoviral infection. Immunostaining showed that the expression levels of hexon protein varied among the cell lines. Normal ceils infected with AxdAdB-3 expressed little hexon protein. The proportion of S-phase cells was （ 39 ± 3 ） % and （ 49 ± 5 ） % in the AxCAlacZ- and AxdAdB-3-infected bladder cancer cells, respectively. AxdAdB-3 effectively induced S-phase entry of cell cycle （ P 〈 0.05 ）. AxdAdB-3 combined with gemcitabine significantly inhibited the growth of bladder cancer cell lines. In vivo, the mean weight of the bladder tumors in mice treated with intravesical instillation of AxCAIacZ,gemcitabine, AxdAdB-3, and AxdAdB-3 ＋ gemcitabine were 400.6, 126.4, 82.0, 40.4 mg, respectively. Either AxdAdB-3 （ P 〈 0. 0001 ） and gemcitabine （ P 〈 0.0001 ） suppressed the tumor growth in n

关 键 词：膀胱肿瘤 溶瘤病毒 吉西他滨 化学疗法 肿瘤 局部灌注 

分 类 号：R737[医药卫生—肿瘤]