永生化山羊子宫内膜上皮细胞系的建立及其生物学特性

为建立永生化山羊子宫内膜上皮细胞系并研究其生物学特性,将人端粒酶逆转录酶催化亚单位(Human telomerase reverse transcrip tase,hTERT)导入原代培养的山羊子宫内膜上皮细胞(Endometrium epithelial cells,EECs),经G418筛选培养,以获得一株永生化山羊子宫内膜上皮细胞(hTERT-EECs);利用流式细胞仪检测其生长周期、凋亡情况及倍体情况;利用MTT法检测其血清依赖性;软琼脂克隆形成试验检测其在半固体培养基中的克隆形成能力;裸鼠致瘤实验检测其成瘤性。结果显示:hTERT-EECs处于S期的细胞(43.2%±2.3%)大于2代EECs(38.7%±1.2%),差异显著(P0.05);hTERT-EECs的凋亡率(1.1%±0.2%)明显低于2代EECs(3.6%±0.5%)(P0.05);倍体分析结果表明hTERT-EECs为二倍体细胞;血清依赖性检测证明hTERT-EECs仍有血清依赖性;软琼脂中无克隆形成能力;对于裸鼠无致瘤性。证明hTERT-EECs具有更强的生长活性,基本属于正常细胞,不具有潜在致瘤性。

Establishment of goat immortalised endometrium epithelial cell line and study on its biological characteristics

To establish immortalised endometrium epithelial cell line (EECs) of goat and study its biological characteristics,primary cultured goat EECs were transfected with human telomerase reverse transcrip tase (hTERT) and obtained were screened by G418.The growth cycle,apoptosis status,and ploidy of hTERT-EECs were detected by flow cytometry;The serum requirement of hTERT-EECs were studied by MTT method;The clone formation ability of hTERT-EECs in semi- solid culture medium was detected by soft agar clone formation test;Its tumorigenicity was observed in nude mice.The results showed:hTERT-EECs in the S phase (43.2%±2.3%) were significantly larger than the second generation EECs (38.7%±1.2%) (P<0.05).The apoptosis rate of hTERT-EECs (1.1%±0.2%) were significantly less than second generations EECs (3.6%±0.5%)(P<0.05);Ploidy analysis showed that hTERT-EECs were diploid;Serum requirement of hTERT-EECs was persistent;The ability of colony forming in soft agar was lost and it didn''t cause tumor in nude mouse.In,conclusion,the hTERT-EECs establish in this study had stronger growth activity without tumorigenicity.