摘要目的 探讨缓激肽B2受体(bradykinin B2 receptor,B2R)对人绒毛外滋养细胞增殖和功能的影响及其可能机制. 方法 人绒毛外滋养细胞(HTR-8/SVneo细胞)分为4组:空载体质粒组转染pcDNA-3.1质粒;B2R表达质粒组转染pcDNA3.1-B2R质粒;小干扰RNA(small interference RNA,siRNA)阴性对照组转染siRNA阴性对照序列;B2R特异性siRNA组转染B2R特异性siRNA序列.采用实时荧光定量逆转录聚合酶链反应技术和蛋白质印迹技术检测转染后细胞中B2R以及基质金属蛋白酶-2、基质金属蛋白酶-9、细胞周期蛋白-1和血管内皮生长因子-A mRNA及蛋白的表达变化.采用细胞计数试剂盒-8及流式细胞术检测细胞活性以及细胞周期;细胞迁移实验和侵袭实验检测细胞迁移和侵袭能力;细胞成环实验检测细胞成血管能力.采用t检验进行统计学分析. 结果 (1)与空载体质粒组相比,B2R表达质粒组细胞中B2R mRNA(5.06±0.49与1.00±0.28,t=7.226,P=0.002)及蛋白表达水平升高(1.34±0.07与1.00±0.05,t=3.727,P=0.006);与siRNA阴性对照组相比,B2R特异性siRNA组细胞中B2R mRNA(0.34±0.05与1.00±0.17,t=3.667,P=0.021)及蛋白表达水平降低(0.74±0.03与1.00±0.05,t=4.097,P=0.006).(2)与空载体质粒组相比,B2R表达质粒组细胞增殖活性升高(1.50±0.03与1.34±0.04),使细胞周期由G0/G1期向S期转变;与siRNA阴性对照组相比,B2R特异性siRNA组细胞增殖活性降低(1.06±0.04与1.20±0.02),并使细胞周期停滞于G0/G1期;差异均有统计学意义(P值均＜ 0.05).(3)与空载体质粒组相比,B2R表达质粒组细胞迁移距离[(80.67±0.33)与(41.33±5.24)μm]、穿膜细胞数[(360.70±12.33)与(268.70±14.45)个]及细胞成环数增加[(28.20±2.47)与(14.00±1.67)个];与siRNA阴性对照组相比,B2R特异性siRNA组细胞迁移距离[(56.00±3.51)与(87.00±1.53)μm]、穿膜细胞数[(143.30±12.91)与(252.30±17.07)个]及细胞成环数减少[(6.25±1.49)与(15.75±2.02)个];差异均有统计学意义(P值均＜0.05).(4)基质金属蛋白酶-2和基质金属蛋白酶-9 mRNA,以及细胞周期蛋白-1和血管内皮生长因子-AmRNA及蛋白表达水平在B2R表达质粒组高于空载体质粒组,在B2R特异性siRNA组低于siRNA阴性对照组,差异均有统计学意义(P值均＜ 0.05). 结论 B2R可能通过促进基质金属蛋白酶-2、基质金属蛋白酶-9、细胞周期蛋白-1和血管内皮生长因子-A的表达,从而增强人绒毛外滋养细胞活性,以及迁移、侵袭及成环能力.

abstractsObjective To investigate the effects and its mechanisms of bradykinin B2 receptor (B2R) on the growth and function of human extravillous trophoblast cells (HTR-8/SVneo cells).Methods B2R expression plasmid (pcDNA3.1-B2R) was constructed and B2R-specific small interfering RNA (siRNA) was synthesized.HTR-8/SVneo cells were divided into four groups and transfected with pcDNA-3.1 (blank plasmid group),pcDNA3.1-B2R (B2R expression plasmid group),siRNA negative control and B2R-specific siRNA,respectively.Quantitative real-time reverse transcription-polymerase chain reaction and Western blot were used to detect the changes in the expression of B2R,matrix metalloproteinase-2,matrix metalloproteinase-9,cyclin D1 and vascular endothelial growth factor-A at both mRNA and protein levels in HTR-8/SVneo cells.Cell counting kit-8 and flow cytometry were used to detect cell activity and cell cycle,respectively.Cell migration assay and cell invasion assay were used to detect cell migration and invasion,respectively.Tube formation assay was used to evaluate the tube formation abilities of HTR-8/SVneo cells.All data were analyzed with t test.Results (1) Compared with the blank plasmid group,expression of B2R in HTR-8/SVneo cells in the B2R expression plasmid group were significantly increased at both mRNA (5.06±0.49 vs 1.00±0.28,t=7.226,P=0.002) and protein levels (1.34 ± 0.07 vs 1.00± 0.05,t=3.727,P=0.006).And the expression of B2R in HTR 8/SVneo cells transfected with B2R-specific siRNA were significantly reduced at both mRNA (0.34±0.05 vs 1.00±0.17,t=3.667,P=0.021) and protein levels (0.74±0.03 vs 1.00±0.05,t=4.097,P=0.006) comparing with the siRNA negative control group.(2) Compared with the blank plasmid group,HTR-8/SVneo cells being transfected with B2R expression plasmid showed a higher proliferation activity (1.50 ±0.03 vs 1.34± 0.04) promoting G0/G1 to S phase transition;compared with the siRNA negative control group,B2R-specific siRNA inhibited the proliferation of HTR-8/SVneo cells (1.06 ± 0.04 vs 1.20± 0.02) and arrested the cell cycle at G0/G 1 phase (all P＜0.05).(3) Compared with the blank plasmid group,B2R expression plasmid significantly increased the HTR-8/SVneo cell migration distance [(80.67±0.33) vs (41.33±5.24) μm],the number of cells penetrating matrigel gel (360.70 ±12.33 vs 268.70 ±14.45) and the number of cells having tube-like structures (28.20 ± 2.47 vs 14.00± 1.67),while significantly decrease was shown in these three parameters in B2R-specific siRNA group comparing with the siRNA negative control group [HTR-8/SVneo cell migration distance:(56.00±3.51) vs (87.00±1.53) μ m,number of cells penetrating matrigel gel:143.30± 12.91 vs 252.30± 17.07;number of tube-like structures:6.25±1.49 vs 15.75 ±2.02;all P＜0.05].(4) Expression of matrix metalloproteinase-2 and matrix metalloproteinase-9 at mRNA level,and expression of cyclin D1 and vascular endothelial growth factor-A increased in the B2R expression plasmid group than in the blank plasmid group,and decreased in the B2R-specific siRNA group than in the siRNA negative control group at both mRNA and protein levels (all P＜0.05).Conclusions B2R might enhance the activity,migration,invasion and tube formation ability of human extravillous trophoblast cells through promoting the expression of matrix metalloproteinase-2,matrix metalloproteinase-9,cyclin D1 and vascular endothelial growth factor-A.