山羊子宫内膜上皮细胞转染pCI-neo-hTERT质粒后的永生化

Immortalization of goat endometrial epithelial cells by telomerase reverse transcriptase transfection

为获得大量的、具有高度同一性和表型功能正常的山羊子宫内膜上皮细胞(EEC),本试验将含人端粒酶逆转录酶(hTERT)基因的真核表达质粒pCI-neo-hT ERT导入原代山羊EEC中,筛选稳定转染的细胞克隆,进行表型鉴定,利用免疫细胞化学方法检测hTERT导入后细胞端粒酶活性的表达情况,探讨转染后细胞的生长特性。结果显示,转染后获得的阳性克隆细胞及其传代细胞呈明显铺路石状,与原代细胞形态一致,具有接触抑制性;角蛋白检测阳性;细胞具有较高的端粒酶活性,目前已传至50代仍稳定增殖;100nmol/L的雌二醇(E2)可促进该细胞的增殖;50,100nmol/L的孕酮(P4)可明显抑制该细胞的增殖。这表明hTERT导入山羊EEC后,可使其重建端粒酶活性,从而获得大量的、具有高度同一性和表型正常的细胞。

To obtain a great quantity of goat endometrium epithelial cells（EECs） with highly identity and normal phenotype function, plasmid pCI-neo-hTERT, the human telomerase reverse transcriptase （hTERT） was transfected into goat EECs. Clones resisted G418 were selected and amplified. The phenotype,proliferation and growth characteristic of stably cell clones were detected, also the effect of sex steroids on the proliferation were determined. The results showed that the confluent monolayer of transfected cells looked like cobblestones with contact inhibition. The transfected ceils were positive for cytokeratin and had higher telomerase activity than that of untransfected ceils by immunocytochemieal staining method. It was cultured up to 50 passages and steadily cultured. 100 nmol/L E2 can stimulate the proliferation of transfected EEC;50,100 nmol/L P4 can inhibit the proliferation of transfected EEC. The research indicated that telomerase activity can be activated in goat EEC after hTERT transfected,thus the cells with high identity and normal phenotype were obtained.