哺乳动物雷帕霉素靶蛋白抑制剂temsirolimus对膀胱癌作用的实验研究  

Effect of temsirolimus on bladder cancer cells in vitro and in vivo

作　　者：陈业刚[1] 刘晓强[1] 林英立[1] 吴长利[1] 孙光[1] 

机构地区：[1]天津医科大学第二医院泌尿外科天津市泌尿外科研究所,300211

出　　处：《中华泌尿外科杂志》2011年第9期626-630,共5页Chinese Journal of Urology

摘　　要：目的研究哺乳动物雷帕霉索靶蛋白（mTOR）抑制剂temsirolimus对膀胱癌T24、BIU-87细胞株的作用，探讨以mTOR为靶点治疗膀胱癌的应用前景。方法膀胱癌T24、BIU-87细胞株经不同浓度temsirolimus作用后，采用噻唑盐法检测temsirolimus对细胞株增殖的影响；流式细胞技术检测temsirolimus对细胞周期、细胞凋亡的影响；细胞迁移实验及transwell细胞侵袭实验检测temsirolimus对肿瘤迁移和浸润的影响；蛋白质印迹法检测mTOR的表达情况；制作裸鼠T24细胞株移植瘤模型，检测temsirolimus对移植瘤生长的作用，免疫组化检测移植瘤Ki-67表达情况。结果temsirolimus能够抑制膀胱癌q724、BIU-87细胞株增殖，呈浓度和时间依赖性；temsirolimus能够抑制膀胱癌细胞迁移能力，temsirolimus作用于T24、BIU-87细胞株24h后，0nmol／L组划痕修复率分别为（88．9±14．1）％、（83．6±16．3）％，高于5nmol／L组的（42．7±11．6）％、（36．9±9．7）％，差异有统计学意义（P〈0．05）；temsirolimus能够抑制膀胱癌细胞浸润能力，temsirolimus作用于T24、BIU-87细胞株24h后，0nmol／L组浸润细胞数（26．5±5．8）、（28．2±4．6），高于5nmol／L组的（19．0±3．8）、（21．3±5．1），差异有统计学意义（P〈0．05）；temsirolimus导致膀胱癌细胞周期阻滞于G0／GI期，temsirolimus作用T24、BIU-87细胞株48b后，5nmol／L组G0／G1期细胞分别占（77．46±6．11）％、（73．39±4．94）％，高于0nmol／L组的（65．99±5．01）％、（60．15±3．98）％，差异有统计学意义（P〈0．05）；各组细胞凋亡率差异无统计学意义（P〉0．05）；temsirolimus能够阻断roTOR的磷酸化，T24、BIU-87细胞株0nmol／I。组p-mTOR／β-actin值分别为0．92±0．09、1．01±0．08，明显高于5nmol／L组的0．47±0．05、0．04±0．01，差异有统计学意义（P〈0．05）；temsirolimus能够抑制裸鼠T24细胞株移�Objective To examine the effects of temsirolimus, an inhibitor of mammalian target of rapamycin, on bladder cancer cell lines T24 and BIU-87 in vitro and in vivo for purpose of evaluating the probability of roTOR targeted therapy for bladder cancer. Methods After being treated by a different concentration of temsirolimus, T24 and BIU-87 cells were tested by MTT assay for cell proliferation activity. Cell cycle and apoptosis analysis were performed with flow cytometer. Wound scratch assay was used for cell migration activity and transwell motility assay. Western blot analysis was used to test the mTOR phosphorylation. Subcutaneous inoculation of 6-week-old nude mice was performed using 1 × 10^6 T24 cells in 50% matrigel for both control （n = 10） and temsirolimus （n = 10） groups. The volume of tumors was examined and then the expression of Ki-67 was detected by immnnobistocbemistry. Results Temsirolimus significantly inhibited proliferation of T24 and BIU-87 cells in a dose- and time-dependent manner. After administration of temsirolimus on T24 and BIU-87 cell lines for 24 h, the rate of wound healing in 0 nmol/L groups were （88.9±14.1）% and （83.6 ± 16.3）%, which were higher than in the 5 nmol/L groups, which were （42.7 ±11.6） % and （36.9 ± 9.7 ） % （ P 〈 0.05 ）. In the transwell motility assay, the number of cells in the 0 nmol/L group was 26.5 ± 5.8 and 28.2 ± 4.6, which was higher than in the 5 nmol/L group （ 19.0 ± 3.8 and 21.3 ± 5.1, respectively） （P 〈 0.05 ）. When temsirolimus was administered on rF24 and BIU-87 cell lines for 48 h the percentages of cells delayed in phase G0/G1 in 5 nmol/L group were （77.46 ± 6.11）% and （73. 39 ± 4. 94）% respectively, and higher than in the 0 nmol/L group, which were （65.99± 5.01 ） %, （60.15± 3.98） % （ P 〈 0.05 ）. There was no statistically significant difference in the apoptosis rate between the two groups （P 〉 0.05）. In Western blot analysis, the ratios of p-mTOR/β-actin were 0.92 ±0.09 and 1

关 键 词：哺乳动物雷帕霉素靶蛋白 膀胱癌 治疗 

分 类 号：R737.14[医药卫生—肿瘤]