Tham Sin Mun, Esuvaranathan Kesavan, Mahendran Ratha
Department of Surgery, Yong Loo Lin School of Medicine, National University of Singapore, National University Health System.
Department of Surgery, Yong Loo Lin School of Medicine, National University of Singapore, National University Health System; Department of Urology, National University Hospital.
J Vis Exp. 2017 Jan 12(119):55078. doi: 10.3791/55078.
This protocol describes the generation of bladder tumors in female C57BL/6J mice using the murine bladder cancer cell line MB49, which has been modified to secrete human Prostate Specific Antigen (PSA), and the procedure for the confirmation of tumor implantation. In brief, mice are anesthetized using injectable drugs and are made to lay in the dorsal position. Urine is vacated from the bladder and 50 µL of poly-L-lysine (PLL) is slowly instilled at a rate of 10 µL/20 s using a 24 G IV catheter. It is left in the bladder for 20 min by stoppering the catheter. The catheter is removed and PLL is vacated by gentle pressure on the bladder. This is followed by instillation of the murine bladder cancer cell line (1 x 10 cells/50 µL) at a rate of 10 µL/20 s. The catheter is stoppered to prevent premature evacuation. After 1 h, the mice are revived with a reversal drug, and the bladder is vacated. The slow instillation rate is important, as it reduces vesico-ureteral reflux, which can cause tumors to occur in the upper urinary tract and in the kidneys. The cell line should be well re-suspended to reduce clumping of cells, as this can lead to uneven tumor sizes after implantation. This technique induces tumors with high efficiency. Tumor growth is monitored by urinary PSA secretion. PSA marker monitoring is more reliable than ultrasound or fluorescence imaging for the detection of the presence of tumors in the bladder. Tumors in mice generally reach a maximum size that negatively impacts health by about 3 - 4 weeks if left untreated. By monitoring tumor growth, it is possible to differentiate mice that were cured from those that were not successfully implanted with tumors. With only end-point analysis, the latter may be mistakenly assumed to have been cured by therapy.
本方案描述了使用经改造可分泌人前列腺特异性抗原（PSA）的小鼠膀胱癌细胞系MB49在雌性C57BL/6J小鼠中生成膀胱肿瘤的方法，以及肿瘤植入确认程序。简而言之，使用可注射药物麻醉小鼠，使其呈背卧位。排空膀胱尿液，使用24G静脉导管以10µL/20s的速度缓慢滴注50µL聚-L-赖氨酸（PLL）。通过堵塞导管将其留在膀胱中20分钟。移除导管，通过轻轻按压膀胱排空PLL。随后以10µL/20s的速度滴注小鼠膀胱癌细胞系（1×10个细胞/50µL）。堵塞导管以防止过早排空。1小时后，用逆转药物使小鼠苏醒，并排空膀胱。缓慢的滴注速度很重要，因为它可减少膀胱输尿管反流，而膀胱输尿管反流会导致上尿路和肾脏出现肿瘤。细胞系应充分重悬以减少细胞团块，因为这会导致植入后肿瘤大小不均匀。该技术可高效诱导肿瘤。通过尿液PSA分泌监测肿瘤生长。对于检测膀胱中肿瘤的存在，PSA标志物监测比超声或荧光成像更可靠。如果不进行治疗，小鼠体内的肿瘤通常在约3 - 4周内达到对健康产生负面影响的最大尺寸。通过监测肿瘤生长，可以区分治愈的小鼠和未成功植入肿瘤的小鼠。仅进行终点分析时，可能会错误地认为后者已通过治疗治愈。
