CAJ | 학술논문

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/ml)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-time PCR及Western印迹检测Netrin-1 mRNA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-1 mRNA水平较对照组增加约40.38倍,两组间差异有统计学意义(P<0.05).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-1 mRNA表达,Netrin-1/Neogenin通路参与了TEV-1细胞凋亡过程.
목적관찰가용성Netrin-1수체대인자양세포증식조망적영향,탐구Netrin-1/Neogenin통로재자양세포조망과정중적궤제.방법행조잉기자양세포주TEV-1배양,세포예처리24h후,개용함불동농도Neogenin적배양액,이농도위0ng/ml적배양액위대조조,기여불동농도Neogenin(1、5、10、50ng/ml)간예적배양액균위실험조.응용새서람(MTT)비색법、류식세포술(FCM)검측세포증식지수、조망솔;채용세포면역형광법검측Netrin-1단백재TEV-1세포내적표체,real-time PCR급Western인적검측Netrin-1 mRNA급Netrin-1단백표체정황.결과불동농도적Neogenin대TEV-1세포증식무명현영향,단TEV-1세포조망솔수 Neogenin농도적증가이증가.Netrin-1표체우자양세포포질.실험조당 Neogenin농도체도50ng/ml시,TEV-1세포표체Netrin-1 mRNA수평교대조조증가약40.38배,량조간차이유통계학의의(P<0.05).Netrin-1단백표체정황수Neogenin농도영향불명현(P>0.05).결론가용성Netrin-1수체능촉진TEV-1세포조망급Netrin-1 mRNA표체,Netrin-1/Neogenin통로삼여료TEV-1세포조망과정.
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 cel s were treated with different concentrations of Neogenin (0, 1, 5, 10 and 50ng/ml). After 24h of treatment cel viability was measured by MTT assay;cel apoptosis were assessed by flow cytometry (FCM) assay;the expression of Netrin-1 in TEV-1 cel s was observed by indirect cel ular 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 cel s was independent of Neogenin. Meanwhile, cel 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 cel s 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 cel 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 cel apoptosis in TEV-1 cel s, which indicates that Netrin-1/Neogenin pathway may play a role during regulating cell apoptosis of trophoblasts.