聚肌胞对人早孕绒毛外滋养细胞TLR表达的调节作用.docVIP

聚肌胞对人早孕绒毛外滋养细胞TLR3表达的调节作用 作者：王菊1,钟刚2 作者单位：1 518106 广东深圳，深圳市光明新区公明人民医院妇科 2 广东深圳，华中科技大学同济医学院附属同济医院 【摘要】目的 通过体外实验研究Toll样受体3(TLR3)在人早孕绒毛外滋养细胞株TEV-1的表达及聚肌胞PolyI:C对TLR3 mRNA和蛋白的调节作用。方法 体外培养TEV-1细胞;用不同时间及浓度聚肌胞刺激TEV-1细胞后RT-PCR和流式细胞仪检测其TLR3 mRNA和蛋白的表达变化;免疫细胞化学SP染色法检测TLR3蛋白的定位表达。结果 (1)PolyI:C 刺激可上调TEV-1细胞株TLR3 mRNA的表达(P 0.01)，且与刺激时间和剂量呈正相关。(2) PolyI:C 刺激可上调TEV-1细胞株TLR3蛋白表达的平均荧光强度(P 0.01);但荧光阳性细胞数刺激12h与刺激前比较差异无显著性(P 0.05);刺激24h、48h的荧光阳性细胞数与刺激时间和剂量呈正关(24h P 0.05;48h P 0.01)。(3)免疫细胞化学显示TLR3主要定位于TEV-1细胞株的细胞浆。结论 TEV-1细胞株TLR3的mRNA及蛋白的表达与Poly(I:C) 刺激时间及剂量呈正相关;TLR3主要定位于TEV-1细胞株的细胞浆，而细胞核及细胞膜没有观测到明显表达。提示人早孕绒毛外滋养细胞可能通过细胞浆的TLR3参与早孕母-胎界面的免疫反应。 【关键词】 TLR3;Poly(I:C);TEV-1细胞株;流式细胞仪;平均荧光强度;荧光阳性细胞数;免疫细胞化学 [Abstract] Objective To investigate the regulatory effect of PolyI:C on the expression of TLR3 mRNA and protein in the human extravillous trophoblast cell line (TEV-1)，and whether the regulatory effect depends on PolyI:C s dose and time.Methods Semi-quantitative RT-PCR analysis，flow cytometry and immuo-cytochemistry were used to dedect TLR3 mRNA and protein expression level and location in TEV-I cell line.Results (1)The expression of TLR3 mRNA in TEV-I cell line significantly increased with the PolyI:C s stimulation time and dose increase; compare to without PolyI:C s stimulation(P 0.01).The mean fluorescence intensity of the TLR3 protein in TEV-I cell line increased with the PolyI:C s stimulation time and dose increase; compare to without PolyI:C s stimulation(P 0.01).(2)The number of fluorescence positive cell of the TLR3 protein stimulated 12 hour had no statistics significance(P 0.05); But its increased with the stimulation time and dose increase in 24 and 48 hour intervention(24h P 0.05 ; 48h P 0.01).(3)The Immuocytochemistry result revealed that TLR3 protein was expressed in TEV-I cell line s cytoplasma.However，it s hardly to see the expression in the cellular membrane and nucleus.Conclusion TLR3 protein was expressed in TEV-I cell line and localized to the cytoplas