姜黄素抑制脂多糖诱导的人绒毛外滋养细胞miR-155表达及细胞凋亡

发布时间：2018-01-07 12:38

本文关键词：姜黄素抑制脂多糖诱导的人绒毛外滋养细胞miR-155表达及细胞凋亡　出处：《南京中医药大学》2016年硕士论文　论文类型：学位论文

【摘要】：[研究背景及目的]人绒毛外滋养细胞(HTR-8/SVne o)过度凋亡是滋养细胞侵入能力下降的重要因素,是引起子痫前期的原因之一。既往研究发现,PE发病中Tall Like Receptor4 (TLR4)炎症通路激活起重要作用。本实验考察姜黄素对于脂多糖(LPS)诱导的人绒毛外滋养细胞过度凋亡的干预作用及其机制。[研究方法]体外培养人绒毛外滋养细胞(HTR-8/SVneo细胞株),分为姜黄素干预组(12.5,25,50μmol/L)、阳性对照组(LPS组、重组腺病毒Ad-miR-155组)、阴性对照组。采用real-time PCR检测不同组别细胞内的miR-155基因表达量；报告基因检测NF-κB信号通路活性；Transwell检测绒毛外滋养细胞侵袭能力；ELISA定量检测不同组别的细胞凋亡情况以及用BCA试剂盒蛋白定量,Western blotting法分析不同组别细胞内的NF-κB (p65)蛋白表达情况。[研究结果11、real-time PCR结果显示LPS处理可上调细胞内miR-155的表达；NF-κ,B报告基因和Western blotting结果显示,LPS激活了绒毛外滋养细胞中NF-kB炎症通路。2、ELISA细胞凋亡检测和Transwell侵袭试验证明,LPS通过miR-155诱导绒毛外滋养细胞凋亡过度,导致其侵袭力下降。3、Western blotting和其q-PCR结果显示,不同剂量的姜黄素预处理组(12.5,25,50μmol/ L)均可降低细胞内NF-κB (p65)蛋白含量,当姜黄素浓度为12.5pmol/L时降低作用最显著；NF-κB报告基因结果提示姜黄素可抑制NF-κB的活性,并呈剂量依赖性。细胞凋亡检测结果表明与对照组相比,LPS处理可诱导细胞凋亡,而姜黄素(12.5,25,50 μmol/L)预处理后可不同程度地抑制细胞凋亡；细胞侵袭试验提示姜黄素(12.5,25,50 μmol/L)预处理后可不同程度地抑制人绒毛外滋养细胞迁移能力下降。[研究结论1LPS通过miR-155促进滋养细胞凋亡,姜黄素预处理可以通过抑制TLR4通路激活,使NF-κB (p65)蛋白下调,下调LPS诱导的miR-155表达水平,从而抑制滋养细胞过度凋亡和侵袭能力下降。
[Abstract]:Background and purpose: human extravillous trophoblast cells (HTR-8/SVne o) is an important factor in the excessive apoptosis of trophoblast invasion ability decreased, which is one of reasons of preeclampsia. Previous studies have found that the incidence of PE in Tall Like Receptor4 (TLR4) inflammatory pathway activation plays an important role. The effects of curcumin on lipopolysaccharide (LPS) cultured human trophoblast cells cultured in vitro. The effect and mechanism of research methods] excessive apoptosis induced human extravillous trophoblast cells (HTR-8/SVneo cells), divided into curcumin intervention group (12.5,25,50 mol/L), positive control group (LPS group, recombinant adenovirus Ad-miR-155 group), negative control group using real-time PCR detection. The expression of miR-155 gene in different groups of cells; report gene detection of NF- kappa B signaling pathway activity; the invasion ability of trophoblast cells to detect Transwell ELISA of different groups; quantitative detection The other cell apoptosis and BCA protein quantitative kit, analysis of different groups of cells within the NF- kappa B Western blotting (p65) protein expression. The results of real-time PCR 11, the results showed that LPS treatment may upregulate the expression of miR-155 in cells; NF- K, B report gene and Western blotting showed that LPS activated extravillous trophoblast cells in NF-kB inflammatory pathways.2, ELISA apoptosis assay and Transwell invasion assay proved that LPS induced by miR-155 trophoblast cell apoptosis, leading to its invasion by.3, Western blotting and the q-PCR results showed that curcumin pretreatment group with different doses (12.5,25,50 mol/ L) can reduce intracellular NF- kappa B (p65) protein content, when curcumin concentration of 12.5pmol/L decreased most significantly; NF- kappa B reporter gene showed that curcumin can inhibit the activity of NF- K B, and dose according to Lai. Cell apoptosis detection results show that compared with the control group, LPS treatment can induce cell apoptosis, and curcumin (12.5,25,50 mol/L) after pretreatment can inhibit cell apoptosis; cell invasion test showed that curcumin (12.5,25,50 mol/L) after pretreatment can inhibit human extravillous trophoblast cell migration ability the conclusion of the study. 1LPS miR-155 by promoting apoptosis of trophoblast cells, curcumin pretreatment can be activated by inhibiting the TLR4 pathway, NF- kappa B (p65) protein expression, downregulation of LPS induced miR-155 expression, thus inhibiting the invasion and apoptosis of trophoblast cells ability decreased.

【学位授予单位】：南京中医药大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R714.244

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