CAJ | 학술논문

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AxCAlacZ感染人膀胱癌细胞株YTS-1、T24、5637、KK47和正常细胞株HCV29、WI38,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48 h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40.4 mg(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.
목적 탐토쌍돌변용류선병독AxdAdB-3연합길서타빈치료라서원위방광암적효과.방법 이함유LacZ기인적선병독AxCAlacZ감염인방광암세포주YTS-1、T24、5637、KK47화정상세포주HCV29、WI38,용5-추-4-록-3-신타-β-D-반유당감염색,검측불동세포주대선병독적역감성.이AxCAlacZ화AxdAdB-3감염상술세포,면역조화염색검측불동세포중선병독결구단백적표체.채용류식세포술검측경선병독감염후YTS-1세포중S기세포적비례.채용세포계수합8법검측AxdAdB-3화(혹)길서타빈대불동방광암세포적살상능력.건립라서원위방광종류모형,관찰방광내관주AxdAdB-3화(혹)길서타빈대방광종류적생장억제효과.결과 불동적세포주대선병독적역감성불동,5637화KK47세포주역감성교강,이YTS-1화T24세포역감성교차.면역조화염색현시,AxdAdB-3지선택성지재종류세포내복제,이재정상세포내불복제.AxCAlacZ혹AxdAdB-3감염YTS-1세포48 h후,S기세포적비례분별위(39±3)％화(49±5)％(P＜0.05).체외화체내실험적결과균현시,AxdAdB-3화길서타빈연합사용비단일사용가경가유효억제방광암세포적생장,기중AxCAlacZ조、길서타빈조、AxdAdB-3조화길서타빈+AxdAdB-3조적평균류중분별위400.6、126.4、82.0화40.4 mg(P＜0.001).결론 AxdAdB-3연합길서타빈방광내관주가유효치료라서방광원위종류,치득진일보개전상관림상연구.
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-β-galactoside (X-Gal).Immunostaining against adenoviral hexon protein was performed to determine the selective replication of AxdAdB-3 in the cancer cells.Flow cytometry was used to determine the YTS-1 cells in S phase of cell cycle after adenovirus infection.Cell viability after AxdAdB-3 and/or gemcitabine was measured by CCK-8 assay.Orthotopic 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 cells 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 cells 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 rumors in mice treated with intravesical instillation of AxCAlacZ,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 nude mice,and the combination therapy reduced tumors more effectively than either AxdAdB-3 (P ＜ 0.0001) or gemcitabine (P ＜ 0.0001) alone.Conclusions Intravesical instillation therapy with AxdAdB-3 in combination with gemcitabine can effectively inhibit the orthotopic bladder cancer in nude mouse,and further relevant clinical studies are guaranteed.