轴突导向因子netrin-1在滋养层细胞侵袭中的作用

摘要

目的 探讨轴突导向因子netrin-1在滋养层细胞侵袭中的作用及其机制.方法 采用RT-PCR技术检测绒毛膜外滋养细胞株TEV-1中netrin-1的6种受体(UNCSA、UNCSB、UNCSC、UNCSD、DCC和neogenin)mRNA的表达.将培养的细胞按照加入netfin-1蛋白浓度的不同分为10μg/L组、50μg/L组、100 μg/L组、500 μg/L组、1000μg/L组、5000μg/L组和阴性对照组(netrin-1的浓度为0 μg/L)共7组,分别用细胞计数试剂盒8检测各组细胞的增殖能力[以吸光度(A)值表示],体外侵袭实验检测各组细胞的侵袭能力.结果 (1)RT-PCR技术检测结果显示,netrin-1的6种受体中,仅检测到neogenin和UNC5B mRNA的表达.(2)培养72 h后,10μg/L组、50μg/L组、100μg/L组、500μg/L组、1000μg/L组和5000 μg/L组细胞的A值分别为1.55±0.29、1.72±0. 31、2. 15±0.35、1.42±0.25、1.50 ±0.27和1.38±0.23,分别与阴性对照组(1.00±0.16)比较,差异均有统计学意义(P<0.05).(3)培养6 h后,10μg/L组、50μg/L组、100μg/L组、500μg/L组、1000μg/L组和5000 μg/L组细胞的穿膜细胞数分别为(41±4)、(47±5)、(55±6)、(44 ±5)、(43±5)和(42±5)个,分别与阴性对照组[(30±4)个]比较,差异均有统计学意义(P<0.05).(4)neogenin mRNA的表达水平,10μg/L组、50μg/L组、100μg/L组、500μg/L组、1000μg/L组和5000 μg/L组分别为1.50±0.16、1.83±0.19、2.24±0.25、2.12±0.24、2.12±0.23和2.13±0.23,分别与阴性对照组(1.00±0.11)比较,差异均有统计学意义(P<0.05);10 μg/L组与50μg/L组比较,50μg/L组与100μg/L组比较,差异也均有统计学意义(P<0.05);100 μg/L组与500 μg/L组、1000 μg/L组和5000μg/L组之间两两比较,差异均无统计学意义(P>0.05).(5)UNCSB mRNA的表达水平,10μg/L组、50μg/L组、100 μg/L组、500 μg/L组、1000 μg/L组和5000 μg/L组分别为1.09±0.11、1.47±0.14、1.61±0.16、1.85±0.19、2.21±0.21和2.42±0.23,分别与阴性对照组(1.00 ±0.07)比较,差异均有统计学意义(P<0.05);各组之间分别比较,差异也均有统计学意义(P<0.05).结论 netrin-1能促进入绒毛膜外滋养细胞的增殖和侵袭能力,这种促进作用受到其受体neogenin及UNCSB的调控.%Objective To investigate mechanism of netrin-1 regulating invasion of extra villous trophoblasts. Methods RT-PCR was used to detect six receptors expression including UNC5A, UNC5B, UNC5C, UNC5D, DCC and neogenin in extra villous trophoblast cell line TEV-1. The TEV-1 cells were cultured and devided into seven groups according to the concentration of netrin-1 adding into the medium, which include 10 μg/L, 50 μg/L, 100 μg/L, 500 μg/L, 1000 μg/L, 5000 μg/L and the control(the concentration of netrin-1 was 0 μg/L) groups. The proliferation and invasion of TEV-1 induced by netrin-1 were determined by CCK-8 assay and transwell invasion assay respectively. Results (1) Only neogenin and UNC5B were found to be expressed on TEV-1 by RT-PCR method. (2) In CCK-8 proliferation assay, after 72 hours culture, the proliferation of TEV-1 were 1.55 ±0.29 in 10 μg/L, 1.72±0. 31 in 50 μg/L, 2.15 ±0.35 in 100 μg/L, 1.42 ±0. 25 in 500 μg/L, 1.50±0. 27 in 1000 μg/L, and 1.38±0.23 in 5000 μg/L group, which were all higher than 1.00 ± 0.16 in control group significantly ( P<0.05 ). (3) In matfigel invasion assay, after 6 hours culture, the number of the trans-membrane cells in various netrin-1 group, including 41 ±4 in 10 μg/L, 47 ±5 in 50 μg/L, 55±6 in 100 μg/L, 44 3=5 in 500 μg/L, 43±5 in 1000 μg/L and 42 ±5 in 5000 μg/L group, were all higher than 30 ±4 in control group with statistical significance( P<0.05 ). (4) The fold changes of neogenin were 1.50 ± 0.16 in 10 μg/L, 1.83 ± 0.19 in 50 μg/L, 2.24 ± 0.25 in 100 μg/L, 2.12 ±0.24 in 500 μg/L, 2.12±0.23 in 1000 μg/L and 2.13 ± O. 23 in 5000 μg/L group, which were all higher than 1.00 ±0.11 in control group significantly( P <0.05 ). There were significant difference between group 10 μg/L and 50 μg/L, group 50 μg/L and 100 μg/L (P < 0.05). There were no significant difference between group 100 μg/L and 500 μg/L, group 1000 μg/L and 5000 μg/L (P>0.05). (5) The fold changes of UNC5 B 1.09 ± 0.11 in 10 μg/L, 1.47±0.14 in 50 μg/ L, 1.61 ±0.16 in 100 μg/L, 1.85±0.19 in 500 μg/L, 2.21±0.21 in 1000 μg/L and 2.42±0.23 in 5000 μg/L group, were all higher significantly when compared with 1.00 ± 0.07 in control group (P<0.05 ). There were significant difference between all groups ( P<0.05). Conclusion Netfin-1 can promote the potential of proliferation and invasion of extravillous trophohlasts in vitro through its receptors including neogenin and UNC5 B.