人类输卵管上皮永生化细胞系的建立及鉴定

Immortalization of human fallopian tube epithelial cells

背景与目的:最近研究表明卵巢高级别浆液性癌可能起源于输卵管上皮。本课题通过基因沉默和端粒酶导入技术将原代输卵管上皮细胞永生化,为以后建立恶性转化细胞系和卵巢癌动物模型奠定基础。方法:分离并培养输卵管上皮细胞,导入p53和pRb shRNA结合hTERTcDNA,建立p53和pRb基因同时沉默并过表达hTERT的永生化细胞系,并对永生化细胞系进行连续传代、沉默基因的蛋白质印迹法(Western blot)测定、SA-β-gal染色和非停泊生长实验及体内致瘤活性鉴定。结果:我们建立了稳定表达p53、pRbshRNA和hTERT的永生化输卵管上皮细胞系:FTE248116/p53i+pRbi+hTERT及FTE312249/p53i+pRbi+hTERT。结论:可以通过导入端粒酶基因及敲除抑癌基因p53和pRb来建立永生化细胞系,并有望于构建恶性转化细胞系和人类高级别浆液性卵巢癌动物模型。

Background and purpose： Recent researches indicate that high grade serous ovarian carcinoma is derived from fallopian tube epithelial malignancy. In this study, we used primary fallopian tube epithelium to establish the immortalized cell lines by silencing of tumor suppression genes and introduction of hTERT, in order to further build malignant cell lines and ovarian cancer animal models. Methods： Primary fallopian tube epithelia were isolated and transfected with a plasmid containing pRb and p53 shRNAs combined with hTERT to establish immortalized cell lines. The resulting cells were validated through examining cell population doublings, p53 and pRb expression, SA- ^-gal activity; anchorage-independent growth and in vivo tumorgenesis. Results： We successfully established two immortalized fallopian tube epithelial cell lines： FTE248116/p53i＋pRbi＋hTERT, FTE312249/p53i＋pRbi＋hTERT through silencing of p53 and pRb and introduction of hTERT simultaneously. Conclusion： The establishment of immortalized cell lines can be accomplished by introduction of p53 and Rb shRNA and overexpression of hTERT, and these cells may be used to establish the transformed cell lines and ovarian cancer animal models.