[Conditionally immortalized human colorectal crypt cell line].
To establish human colorectal crypt cell line.



Colorectal crypt cells were separated from human fetal gut by dispase I digestion, AKP-negative cells from fetal colorectal crypt were collected and cultured on Matrigel matrix. Subsequently the primary cultured cells were transfected with recombinant retrovirus containing human telomerase reverse transcriptase (hTERT) and simian virus 40 large T antigen (SV40 LT) in 48 h. The characterization of immortalized cells was identified after the transfection and cells were screened with antibiotics for 12 approximately 16 weeks and expanded.



Mucin, cytokeratin-pan, 8, 19 were presented in immortalized cells by immunohistochemical staining; ectopic expressions of both hTERT and SV40 LT were also found in immortalized cells by Western blotting. Agarose electrophoresis showed that the cells expressed Musashi-1 mRNA. No evidence of carcinogenesis was found in nude mouse experiment and soft-agarose cloning test.



The immortalized human colorectal crypt cells were characterized and the established cell line may be an ideal target for carcinogenesis study in vitro.
Zhu
Yong-liang
YL
The Second Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310009, China.
Zhong
Xian
X
Zheng
Shu
S
D002461:Cell Line, Transformed; D003106:Colon / Q000166:cytology*; D004268:DNA-Binding Proteins; D005333:Fetus; D006801:Humans; D012334:RNA, Neoplasm / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013539:Simian virus 40 / Q000276:immunology; D013234:Stem Cells / Q000166:cytology*; D019098:Telomerase / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection / Q000379:methods
D004268:DNA-Binding Proteins; D012334:RNA, Neoplasm; D019098:Telomerase