In vivo imaging with replication competent retrovirus vector in urologic cancer

1.将携带GFP转基因的慢病毒载体转导至膀胱癌细胞系KU-7和前列腺癌细胞系PC-3，以建立连续表达GFP的人膀胱癌和前列腺癌细胞系。构建KU-7-GFP和PC-3-GFP表达载体，Western blotting检测GFP的表达.（1）皮下模型：将KU-7、KU-7-GFP、PC-3或PC-3-GFP接种于裸鼠皮下，通过Macro-Imaging系统观察GFP表达。所有接种KU-7-GFP或PC-3-GFP的小鼠在1-2周后均识别GFP。(2)膀胱原位移植模型：将5×106 KU-7或KU-7-GFP经尿道注入裸鼠膀胱，观察GFP表达。在所有六只小鼠中，直到八周才检测到GFP表达。另一方面，当从膀胱附近皮下移植KU-7-GFP时，检测到GFP表达。(3)骨转移模型：将PC-3-GFP移植到裸鼠胫骨，通过图像分析系统检测GFP的表达。（4）GFP的长期表达：将KU-7-GFP体外培养至40代，接种于裸鼠皮下。结论：膀胱癌和前列腺癌细胞系的GFP表达能够在体内检测到，原位和转移模型均适用于实验，无需处死小鼠。由于检测GFP表达的灵敏度不是很高，因此在该系统中不会检测到具有复制能力的逆转录病毒载体。因此，需要改进，如光电缆和摄像机，以检测可复制的逆转录病毒载体。

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.