可溶性Netrin-1受体对人滋养细胞株TEV-1增殖凋亡的影响

Effect of soluble Netrin-1 receptor on proliferation and apoptosis of TEV-1 cells

目的观察可溶性Netrin-1受体对人滋养细胞增殖凋亡的影响，探究Netrin-1／Neogenin通路在滋养细胞凋亡过程中的机制。方法行早孕期滋养细胞株TEV-1培养，细胞预处理24h后，改用含不同浓度Neogenin的培养液，以浓度为0ng／ml的培养液为对照组，其余不同浓度Neogenin（1、5、10、50ng／m1）干预的培养液均为实验组。应用噻唑蓝（MTT）比色法、流式细胞术（FCM）检测细胞增殖指数、凋亡率；采用细胞免疫荧光法检测Netrin-1蛋白在TEV-1细胞内的表达，real-timePCR及Western印迹检测Netrin-1mRNA及Netrin-1蛋白表达情况。结果不同浓度的Neogenin对TEV-1细胞增殖无明显影响，但TEV-1细胞凋亡率随Neogenin浓度的增加而增加。Netrin-1表达于滋养细胞胞质。实验组当Neogenin浓度达到50ng／ml时，TEV-1细胞表达Netrin-1mRNA水平较对照组增加约40．38倍，两组间差异有统计学意义（P〈005）。Netrin-1蛋白表达情况受Neogenin浓度影响不明显（P〉0．05）。结论可溶性Netrin-1受体能促进TEV-1细胞凋亡及Netrin-1mRNA表达，Netrin-1／Neogenin通路参与了TEV-1细胞凋亡过程。

Objective To investigate the effect of soluble Netrin-1 receptor on proliferation and apoptosis of human tro- phoblast TEV-1 cells. Methods Cultured TEV-1 cells were treated with different concentrations of Neogenin （0, 1, 5, 10 and 50ng/ml）. After 24h of treatment cell viability was measured by MTT assay; cell apoptosis were assessed by flow cytometry （FCM） assay; the expression of Netrin-1 in TEV-1 cells was observed by indirect cellular immunofluorescence; the mRNA and protein of Netrin-1 were detected by real-time PCR and Western blot, respectively. Results The results of MTT and FCM showed that proliferation of TEV-1 cells was independent of Neogenin. Meanwhile, cell apoptosis increased with the increasing concentrations of Neogenin; the apoptosis rate was 18.70% at 0 ng/ml Neogenin and significantly increased to 56.65% at 50ng/ml Neogenin （P〈 0.01）. Immunoreactivity of Netrin-1 in cytoplasm of TEV-1 cells was increased along with the increasing concentration of Neogenin. Real-time PCR showed the level of Netrin-1 mRNA, increased by 40.38 times compared to control group when TEV-1 cell was exposed to 50ng/ml Neogenin（P〈0.05 ）; and the same tendency was not seen by using Western blot（P 〉0.05）. Con- clusion The soluble Netrin-1 receptor Neogenin enhances Netrin-1 mRNA expression and promotes cell apoptosis in TEV-1 cells, which indicates that Netrin-1/Neogenin pathway may play a role during regulating cell apoptosis of trophoblasts.