本文關(guān)鍵詞：Genistein對人臍靜脈內(nèi)皮細(xì)胞eNOS表達(dá)的影響及其機制　出處：《山西醫(yī)科大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： genistein 內(nèi)皮型一氧化氮合酶 動脈粥樣硬化

【摘要】：目的探討genistein對氧化低密度脂蛋白（ox-LDL）活化的人臍靜脈內(nèi)皮細(xì)胞（HUVECs）中一氧化氮合酶（eNOS）表達(dá)的影響。 方法 1.研究genistein長時間干預(yù)的作用: （1）體外培養(yǎng)人臍靜脈內(nèi)皮細(xì)胞，在ox-LDL（100mg/L）干預(yù)內(nèi)皮細(xì)胞的基礎(chǔ)上，不同濃度的genistein（10nmol/L，50nmol/L，100nmol/L）孵育24h，MTT法檢測HUVECs細(xì)胞活力的變化，逆轉(zhuǎn)錄聚合酶鏈?zhǔn)椒磻?yīng)（RT-PCR）和Western blot觀察genistein對eNOS表達(dá)的影響。 （2）經(jīng)雌激素受體拮抗劑ICI182780（1μ M）或經(jīng)基因轉(zhuǎn)錄阻斷劑-放線菌素D（5mg/L）作用30min，再加入genistein（100nmol/L）作用24h，RT-PCR和Western blot檢測eNOS的表達(dá)。 2.研究genistein短時間干預(yù)的作用：體外培養(yǎng)人臍靜脈內(nèi)皮細(xì)胞，在ox-LDL（100mg/L）干預(yù)內(nèi)皮細(xì)胞的基礎(chǔ)上，，genistein (100nmol/L)分別作用5、10、15、30和60min，Greiss反應(yīng)測定細(xì)胞培養(yǎng)上清中一氧化氮（NO）的含量，RT-PCR檢測HUVECs eNOS mRNA的表達(dá)，Western blot檢測HUVECs eNOS蛋白和磷酸化eNOS（Ser1177）的表達(dá)水平；同時，Western blot觀察PI3K/AKT信號通路抑制劑LY294002和NSC154020干預(yù)后，genistein對HUVECs磷酸化eNOS（Ser1177）表達(dá)的影響。 結(jié)果 1.genistein長時間干預(yù)的作用：(1)MTT結(jié)果顯示：ox-LDL（100mg/L）組細(xì)胞活力明顯受到抑制（P0.05），而genistein則明顯增強內(nèi)皮細(xì)胞增殖活力，隨著genistein濃度的升高（10nmol/L，50nmol/L，100nmol/L），細(xì)胞活力升高越顯著（P0.05）。(2)RT-PCR和Western blot結(jié)果顯示：與正常對照組相比，ox-LDL（100mg/L）組明顯下調(diào)eNOS mRNA和蛋白表達(dá)（P0.05），而genistein明顯上調(diào)eNOS mRNA和蛋白表達(dá)（P0.05）；genistein對eNOS的誘導(dǎo)作用被雌激素受體拮抗劑ICI182780（1μ M）和基因轉(zhuǎn)錄阻斷劑-放線菌素D（5mg/L）明顯的抑制（P0.05）。 2. genistein短時間干預(yù)的作用：HUVECs經(jīng)100nmol/L genistein短時間(5、10、15、30和60min)處理后，培養(yǎng)液NO濃度和磷酸化eNOS(ser1177）表達(dá)水平均明顯高于ox-LDL組（P均0.05)，其中g(shù)enistein15min處理組作用最明顯，然而，eNOS mRNA和非磷酸化eNOS蛋白表達(dá)水平與ox-LDL組比較無統(tǒng)計學(xué)意義(P0.05)；同時，HUVECs經(jīng)PI3K/AKT信號通路抑制劑LY294002和NSC154020干預(yù)后，磷酸化eNOS（Ser1177）表達(dá)明顯低于genistein處理組（P0.05）。 結(jié)論 （1）genistein作用時間比較長（24h）時，通過促進(jìn)eNOS表達(dá)，進(jìn)而提高eNOS的活性，這個過程和雌激素受體介導(dǎo)的基因組途徑密切相關(guān)。 （2）genistein作用時間比較短（60min以內(nèi)）時，genistein對eNOS的誘導(dǎo)作用與其促進(jìn)eNOS（Ser1177）磷酸化進(jìn)而提高eNOS活性密切相關(guān)，PI3K/AKT信號通路在此過程中發(fā)揮重要作用。
[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.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R329

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