正常大肠干细胞的条件永生化

Conditionally immortalized human colorectal crypt cell line

目的 :建立正常人大肠干细胞系。方法 :用中性蛋白酶分级分离法从人胚肠中分离正常大肠干细胞 ,以含端粒酶逆转录酶和 SV4 0大 T抗原的重组逆转录病毒感染 ,对其进行永生化 ;然后 ,对建立的正常大肠干细胞系进行生物学特性鉴定。结果 :用中性蛋白酶分级消化获得的 AKP活性阴性的细胞群生长呈多角形。该细胞群转染含端粒酶逆转录酶和 SV4 0大 T抗原的重组逆转录病毒后 8～ 12周出现上皮样细胞克隆 ,经细胞特性鉴定 :PAS染色黏蛋白阳性 ,上皮细胞角蛋白 pan、CK- 8、CK- 19均阳性 ;用 EL ISA- PCR法检测该细胞第 12代和第 4 3代端粒酶活性 ,分别为 0 .4 3、0 .83;用 Western blot法检测发现该细胞系表达端粒酶和 SV4 0大 T抗原 ;用 RT- PCR检测示该细胞株表达 Musashi- 1m RNA;以 1× 10 6细胞数接种裸鼠 ,观察 4个月无肿瘤形成 ,软琼脂克隆试验培养无转化细胞克隆出现。结论 :建立的正常的大肠干细胞系具有干细胞特征 ,可作为体外研究致癌剂

Objective: To establish human colorectal crypt cell line. Methods: 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 12~16 weeks and expanded. Results: 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. Conclusion: The immortalized human colorectal crypt cells were characterized and the established cell line may be an ideal target for carcinogenesis study in vitro .