Genistein对人脐静脉内皮细胞eNOS表达的影响及其机制

发布时间：2018-01-06 01:11

本文关键词：Genistein对人脐静脉内皮细胞eNOS表达的影响及其机制　出处：《山西医科大学》2012年硕士论文　论文类型：学位论文

【摘要】：目的探讨genistein对氧化低密度脂蛋白（ox-LDL）活化的人脐静脉内皮细胞（HUVECs）中一氧化氮合酶（eNOS）表达的影响。方法 1.研究genistein长时间干预的作用: （1）体外培养人脐静脉内皮细胞，在ox-LDL（100mg/L）干预内皮细胞的基础上，不同浓度的genistein（10nmol/L，50nmol/L，100nmol/L）孵育24h，MTT法检测HUVECs细胞活力的变化，逆转录聚合酶链式反应（RT-PCR）和Western blot观察genistein对eNOS表达的影响。（2）经雌激素受体拮抗剂ICI182780（1μ M）或经基因转录阻断剂-放线菌素D（5mg/L）作用30min，再加入genistein（100nmol/L）作用24h，RT-PCR和Western blot检测eNOS的表达。 2.研究genistein短时间干预的作用：体外培养人脐静脉内皮细胞，在ox-LDL（100mg/L）干预内皮细胞的基础上，，genistein (100nmol/L)分别作用5、10、15、30和60min，Greiss反应测定细胞培养上清中一氧化氮（NO）的含量，RT-PCR检测HUVECs eNOS mRNA的表达，Western blot检测HUVECs eNOS蛋白和磷酸化eNOS（Ser1177）的表达水平；同时，Western blot观察PI3K/AKT信号通路抑制剂LY294002和NSC154020干预后，genistein对HUVECs磷酸化eNOS（Ser1177）表达的影响。结果 1.genistein长时间干预的作用：(1)MTT结果显示：ox-LDL（100mg/L）组细胞活力明显受到抑制（P0.05），而genistein则明显增强内皮细胞增殖活力，随着genistein浓度的升高（10nmol/L，50nmol/L，100nmol/L），细胞活力升高越显著（P0.05）。(2)RT-PCR和Western blot结果显示：与正常对照组相比，ox-LDL（100mg/L）组明显下调eNOS mRNA和蛋白表达（P0.05），而genistein明显上调eNOS mRNA和蛋白表达（P0.05）；genistein对eNOS的诱导作用被雌激素受体拮抗剂ICI182780（1μ M）和基因转录阻断剂-放线菌素D（5mg/L）明显的抑制（P0.05）。 2. genistein短时间干预的作用：HUVECs经100nmol/L genistein短时间(5、10、15、30和60min)处理后，培养液NO浓度和磷酸化eNOS(ser1177）表达水平均明显高于ox-LDL组（P均0.05)，其中genistein15min处理组作用最明显，然而，eNOS mRNA和非磷酸化eNOS蛋白表达水平与ox-LDL组比较无统计学意义(P0.05)；同时，HUVECs经PI3K/AKT信号通路抑制剂LY294002和NSC154020干预后，磷酸化eNOS（Ser1177）表达明显低于genistein处理组（P0.05）。结论（1）genistein作用时间比较长（24h）时，通过促进eNOS表达，进而提高eNOS的活性，这个过程和雌激素受体介导的基因组途径密切相关。（2）genistein作用时间比较短（60min以内）时，genistein对eNOS的诱导作用与其促进eNOS（Ser1177）磷酸化进而提高eNOS活性密切相关，PI3K/AKT信号通路在此过程中发挥重要作用。
[Abstract]:Objective to investigate the effect of genistein on the expression of nitric oxide synthase (NOS) in human umbilical vein endothelial cells (HUVECs) activated by oxidized low density lipoprotein (ox-LDL). Method 1. To study the effect of genistein long-term intervention: Human umbilical vein endothelial cells (HUVECs) were cultured in vitro. On the basis of ox-LDL100 mg / L intervention, different concentrations of genistein(10nmol/L were observed. HUVECs cells were incubated with 50nmol / L and 100nmol / L for 24 h to detect the changes of cell viability. Reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot were used to observe the effect of genistein on eNOS expression. (2) treated with estrogen receptor antagonist (ICI182780(1 渭 M) or gene transcription blocker (actinomycin D 5 mg / L) for 30 min. Then genistein (100 nmol / L) was added to detect the expression of eNOS by RT-PCR and Western blot for 24 h. 2. To study the effects of genistein on human umbilical vein endothelial cells (HUVECs) in vitro, and on the basis of ox-LDL100mg / L) intervention of human umbilical vein endothelial cells (HUVECs). Genistein (100nmol / L) was exposed to 510nmol / L for 1530min and 60min, respectively. The content of nitric oxide (no) in the supernatant of cell culture was determined by Greiss reaction and the expression of HUVECs eNOS mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR). Western blot was used to detect the expression of HUVECs eNOS protein and phosphorylated eNOS-1 Ser1177. At the same time, Western blot was used to observe the effect of LY294002 and NSC154020 on PI3K/AKT signal pathway inhibitor. The effect of genistein on the expression of HUVECs phosphorylated eNOSN Ser1177. Results 1. Genistein intervention for a long time showed that the cell viability was significantly inhibited in the 10 mg / L group (P 0.05). Genistein significantly increased the proliferation activity of endothelial cells, with the increase of genistein concentration, 10 nmol / L ~ (50) nmol / L ~ (10) nmol / L ~ (-1)). The results of RT-PCR and Western blot showed that the higher the cell viability was, the more significant it was compared with the normal control group. In ox-LDL 100mg / L group, the expression of eNOS mRNA and protein was significantly down-regulated (P0.05). However, genistein upregulated the expression of eNOS mRNA and protein (P0.05). The induction of eNOS by genistein was significantly inhibited by estrogen receptor antagonist (ICI182780(1 渭 M) and gene transcriptional blocker (actinomycin DX 5 mg / L) (. P0.05. 2. The role of genistein in short time intervention was 100 nmol / L genistein for a short time. After 30 and 60 min treatment, no concentration and phosphorylated eNOSser1177) in the culture medium were significantly higher than those in the ox-LDL group (P 0.05). The effect of genistein15min was the most obvious, however. The expression level of eNOS mRNA and non-phosphorylated eNOS protein was not significantly higher than that of ox-LDL group (P 0.05). At the same time, HUVECs were treated with PI3K/AKT signal pathway inhibitor LY294002 and NSC154020. The expression of phosphorylated eNOSn Ser1177 was significantly lower than that of genistein treatment group (P 0.05). Conclusion When genistein acted for 24 hours, the activity of eNOS was enhanced by promoting the expression of eNOS. This process is closely related to the estrogen receptor mediated genomic pathway. The action time of genistein was less than 60 min. The induction of eNOS by genistein is closely related to its promotion of eNOS- Ser1177) phosphorylation and thus to the enhancement of eNOS activity. PI3K/AKT signaling pathway plays an important role in this process.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329

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