目的:观察人滋养细胞(TEV-1)经血清剥夺刺激后,VEGF、Netrin-1的表达及分布,同时监测TEV-1增殖、凋亡及管腔形成等生物学行为的变化,探讨血清剥夺对滋养细胞生物行为的影响及其机制。方法:行早孕期滋养细胞株TEV-1培养,并随机分为实验组(无血清培养基处理)及对照组(15%胎牛血清培养基处理),应用MTT比色法、流式细胞术(FCM)检测细胞增殖凋亡情况,并监测管腔形成的变化。同时利用免疫荧光细胞化学染色法及实时定量PCR检测VEGF、Ne-trin-1蛋白表达定位及mRNA变化情况。结果:MTT及FCM检测表明,血清剥夺能抑制TEV-1细胞增殖、诱导TEV-1凋亡。滋养细胞培养24 h后可出现管腔形成现象,且这一现象不受血清剥夺的抑制。VEGF、Netrin-1蛋白均表达于TEV-1细胞胞浆,此外,实验组TEV-1VEGF mRNA与Netrin-1mRNA的表达水平依次为对照组的(0.94±0.02)、(0.62±0.11)倍。结论:血清剥夺后,滋养细胞出现凋亡增加及增殖抑制现象,并且VEGF、Netrin-1表达减少。血清剥夺可能通过影响VEGF、Netrin-1的表达来参与对滋养细胞增殖凋亡的调控。
Objective:To observe the expressions and distributions of vascular endothelial growth factor(VEGF) and Netrin-1 after serum deprivation stimulation of human TEV-1 cells,meanwhile,monitor the changes of biological behaviors including proliferation,apoptosis and tubal formation of TEV-1,explore the effect of serum deprivation on biological behaviors of trophoblast and the related mechanism. Methods:TEV-1 cell line was cultured during the first trimester of pregnancy,then TEV-1 cells were divided into experimental group(including the cells didn't treated with serum medium) and control group(including the cells treated with 15% fetal calf serum medium),MTT assay and flow cytometry(FCM) were used to detect the proliferation and apoptosis of TEV-1 cells,the change of tubal formation was monitored.Meanwhile,Immunofluorescence and real-time qualitative PCR were used to detect the expressions and locations of VEGF and Netrin-1 protein and the changes of their mRNA expressions. Results:The results of MTT and FCM indicated that serum deprivation could inhibit TEV-1 cell proliferation and induce TEV-1 cell apoptosis.After cultivation for 24 hours,tubal formation was detected in TEV-1 cells,and the phenomenon could not be inhibited by serum deprivation.VEGF and Netrin-1 protein both expressed in the cytoplasm of TEV-1 cells,the expression levels of VEGF mRNA and Netrin-1 mRNA in experimental group were(0.94±0.02) times and(0.62±0.11) times than control group. Conclusion: Serum deprivation can inhibit the proliferation and induce the apoptosis of TEV-1 cells,and the expression levels of VEGF and Netrin-1 decreased.Serum deprivation participates in the regulation of proliferation and apoptosis of TEV-1 cells by affecting the expression levels of VEGF and Netrin-1.