In vivo imaging with replication competent retrovirus vector in urologic cancer

1. 用转染GFP基因的透明病毒载体转染膀胱癌细胞株KU-7和前列腺癌细胞株PC-3，建立连续表达GFP的人膀胱癌细胞株和前列腺癌细胞株。构建KU-7-GFP和PC-3-GFP，用免疫印迹法检测GFP的表达。(1)皮下模型：裸鼠皮下接种KU-7、KU-7-GFP、PC-3、PC-3-GFP，通过Macro-Imaging系统观察GFP的表达情况。接种KU-7-GFP或PC-3-GFP的小鼠在1-2周后均能识别出GFP。(2)膀胱原位模型：将KU-7或KU-7-GFP 5×106经尿道注入裸鼠膀胱，观察GFP表达情况。在所有6只小鼠中，直到8周才检测到GFP表达。另一方面，当KU-7-GFP从膀胱附近皮下移植时，检测到GFP的表达。(3)骨转移模型：将PC-3-GFP移植到裸鼠胫骨，通过成像系统检测GFP的表达。(4) GFP的长期表达：KU-7-GFP体外培养至40代，接种于裸鼠皮下。一周后检测GFP表达。总之，膀胱和前列腺癌细胞系的GFP表达在体内均可检测到，原位和转移模型均可用于实验，无需牺牲小鼠。由于检测GFP表达的灵敏度不是很高，因此该系统无法检测到具有复制能力的逆转录病毒载体。因此，需要改进如光缆和照相机来检测复制能力强的逆转录病毒载体。

1. GFP expression in vitroLentivirus vector with GFP transgene was transduced to bladder cancer cell line, KU-7, and prostate cancer cell line, PC-3, to establish human bladder and prostate cancer cell lines that continuously express the GFP. KU-7-GFP and PC-3-GFP were established and GFP expression was detected by the western blotting.2. GFP expression in vivo(1) Subcutaneous model : KU-7, KU-7-GFP, PC-3, or PC-3-GFP was inoculated to nude mouse subcutaneously, and GFP expression was observed by Macro-Imaging system. All mice that inoculated KU-7-GFP or PC-3-GFP were recognized GFP after 1-2 weeks.(2) Bladder orthotopic model : 5×106 of KU-7 or KU-7-GFP was injected to the bladder of nude mouse through urethra and GFP expression was observed. In all six mice, GFP expression was not detected until eight weeks. On the other hand, when KU-7-GFP was transplanted from subcutaneous near bladder, GFP expression was detected.(3) Bone metastasis model : GFP expression was detected by imaging system, when PC-3-GFP was transplanted into the tibia of nude mice.(4)Long term GFP expression : KU-7-GFP was cultured until forty passage in vitro, and was inoculated to subcutaneous in nude mice. GFP expression was detected at one week.In conclusion, GFP expression of bladder and prostate cancer cell lines was able to detect in vivo and both orthotopic and metastatic model were useful for the experiment without sacrificing mice. Because the sensitivity of detection for the expression of GFP is not very high, replication competent retrovirus vector would not be detected in this system. Therefore improvement such as is light cable and camera are needed to detect the replication competent retrovirus vector.