本文選題：硫化氫 + 炔丙基甘氨酸�。� 參考：《蘇州大學(xué)》2012年碩士論文

【摘要】：目的：探討外源性硫化氫對血管平滑肌細(xì)胞內(nèi)吞氧化型低密度脂蛋白的影響，并對其分子學(xué)機(jī)制進(jìn)行初步探討，為動脈粥樣硬化的防治提供新的理論依據(jù)。 方法：采用組織貼塊法從大鼠腹主動脈分離培養(yǎng)血管平滑肌細(xì)胞(vascular smoothmuscle cell，VSMC)。培養(yǎng)基選用含10％胎牛血清(FBS)的DMEM，培養(yǎng)條件為5％CO_2、37℃。培養(yǎng)3～5天有細(xì)胞爬出，7～9天可進(jìn)行第一次傳代。實(shí)驗選用3～4代VSMC。根據(jù)不同檢測方法可分為：①用于MTT檢測細(xì)胞活性的細(xì)胞接種于96孔板，按5×104/mL細(xì)胞密度每孔200μl；②用于油紅O染色、免疫細(xì)胞化學(xué)檢測的細(xì)胞接種于放有小圓玻片的24孔板內(nèi)，按1×105/ml細(xì)胞密度每孔1ml；③.用于免疫印跡檢測的細(xì)胞接種于無菌6孔板中，按1×106/ml細(xì)胞密度，每孔2ml。 實(shí)驗分組： 1、MTT法檢測H2S對VSMC活性影響：分為對照組、NaHS（25、50、100、200、500、1000μmol/L）組； 2、油紅O染色檢測VSMC脂質(zhì)含量與免疫細(xì)胞化學(xué)法檢測CD36、LOX-1受體表達(dá)：分為對照組、ox-LDL（80μg/ml）組、ox-LDL（80μg/ml）+NaHS(50μmol/L)組、ox-LDL（80μg/ml）+PPG(3mmol/L)組； 3、DiI-oxLDL內(nèi)吞的檢測：分為DiI-oxLDL（10μg/ml）組，DiI-oxLDL（10μg/ml）+NaHS(50μmol/L)組，DiI-oxLDL（10μg/ml）+PPG(3mmol/L)組； 4、Western blot測定VSMC的CD36、LOX-1蛋白表達(dá)：分為對照組、ox-LDL（80μg/ml）組、ox-LDL（80μg/ml）+NaHS（25、50、100、200μmol/L）組。 結(jié)果： 1.MTT結(jié)果顯示，NaHS濃度在200μmol/L以下時，對VSMC的活力沒有明顯影響，當(dāng)NaHS濃度增加到500和1000μmol/L后細(xì)胞活力明顯下降。 2.通過油紅O染色觀察外源性H2S（以NaHS為供體）對VSMC脂質(zhì)攝取的影響。結(jié)果發(fā)現(xiàn)，與對照組相比，VSMC與ox-LDL單獨(dú)孵育后胞內(nèi)脂質(zhì)顯著增多，當(dāng)用CSE（胱硫醚-γ-裂解酶）抑制劑PPG作用后，脂質(zhì)沉積更明顯，而H2S處理后的VSMC胞內(nèi)脂質(zhì)明顯減少。 3.用DiI-oxLDL處理VSMC，Confocal觀察VSMC對ox-LDL的內(nèi)吞作用，主要觀察外加DiI-oxLDL在VSMC胞內(nèi)的積聚。結(jié)果觀察到H2S能抑制VSMC對ox-LDL的內(nèi)吞，而PPG則增強(qiáng)這種內(nèi)吞作用，與油紅O染色結(jié)果相符。 4.免疫細(xì)胞化學(xué)法檢測VSMC的CD36、LOX-1受體表達(dá)，對H2S抑制VSMC內(nèi)吞ox-LDL的分子學(xué)機(jī)制進(jìn)行初步探討。結(jié)果顯示，ox-LDL能明顯誘導(dǎo)VSMC的CD36、LOX-1受體表達(dá)，H2S處理后可下調(diào)這兩種受體的表達(dá)，，PPG作用后則使CD36、LOX-1受體表達(dá)上調(diào)。 5.為進(jìn)一步確定H2S抑制VSMC內(nèi)吞脂質(zhì)的機(jī)制是否與其下調(diào)CD36、LOX-1有關(guān)，本實(shí)驗還采用了Western blot方法分析NaHS對VSMC的CD36、LOX-1蛋白表達(dá)的影響。結(jié)果發(fā)現(xiàn)，ox-LDL顯著增強(qiáng)誘導(dǎo)VSMC的CD36、LOX-1蛋白表達(dá)，而H2S對兩種受體蛋白表達(dá)的抑制作用呈劑量依賴性。 結(jié)論: 1.外源性H2S可通過抑制VSMC對ox-LDL的內(nèi)吞減少脂質(zhì)在胞內(nèi)的沉積。 2. H2S能夠抑制VSMC內(nèi)脂質(zhì)沉積，其機(jī)制與其下調(diào)CD36、LOX-1表達(dá)有關(guān)。
[Abstract]:Aim: to investigate the effect of exogenous hydrogen sulfide on endocytosis low density lipoprotein (LDL) in vascular smooth muscle cells (VSMC) and its molecular mechanism in order to provide a new theoretical basis for the prevention and treatment of atherosclerosis.Methods: vascular smooth muscle cells (VSMCs) were isolated from rat abdominal aorta by tissue patch method.The culture medium was DMEM containing 10% fetal bovine serum (FBS).After 3 days of culture, the cells crawled out of the cells for 7 to 9 days for the first passage.Three or four generations of VSMC were used in the experiment.The density of 1 脳 105/ml cells was 1 ml / L ~ 3.The cells for Western blotting were inoculated in the aseptic 6-well plate, and the density of 1 脳 106/ml cells was 2 ml per pore.Experimental groups:4Western blot was used to detect the expression of CD36 blot in VSMC: it was divided into two groups: control group (80 渭 g / ml) of ox-LDL (80 渭 g / ml) NaHS2550100 渭 mol / L) group.Results:The results of 1.MTT showed that when the concentration of nahs was below 200 渭 mol/L, there was no significant effect on the activity of VSMC. When the concentration of NaHS increased to 500 渭 mol/L and 1000 渭 mol/L, the cell viability decreased obviously.2.The effects of exogenous H 2 S (using NaHS as donor) on lipid uptake of VSMC were observed by oil red O staining.The results showed that compared with the control group, the intracellular lipids increased significantly after ox-LDL was incubated alone, and the lipid deposition was more obvious when treated with CSE- 緯 -lyase inhibitor PPG, while the intracellular lipid of VSMC treated with H2S was significantly decreased.3.The endocytosis of VSMC on ox-LDL was observed with DiI-oxLDL treatment, and the accumulation of DiI-oxLDL in VSMC cells was observed.Results it was observed that H 2S could inhibit the endocytosis of ox-LDL by VSMC, while PPG enhanced the endocytosis, which was consistent with the results of oil red O staining.4.The expression of CD36 LOX-1 receptor in VSMC was detected by immunocytochemistry, and the molecular mechanism of H2S inhibiting endocytosis of ox-LDL in VSMC was preliminarily discussed.The results showed that ox-LDL could significantly induce the expression of LOX-1 receptor in VSMC. H2S could down-regulate the expression of these two receptors and up-regulate the expression of LOX-1 receptor after PPG treatment.5.In order to further determine whether the mechanism of inhibiting VSMC endocytosis by H2S is related to its down-regulation of CD36nLOX-1, the effect of NaHS on the expression of VSMC CD36 LOX-1 protein was analyzed by Western blot method.The results showed that ox-LDL significantly enhanced the expression of CD36 / LOX-1 protein induced by VSMC, while H2S inhibited the expression of two receptor proteins in a dose-dependent manner.Conclusion:1.Exogenous H 2S can reduce lipid deposition in ox-LDL by inhibiting the endocytosis of ox-LDL by VSMC.2.H2S can inhibit lipid deposition in VSMC, and its mechanism is related to its down-regulation of CD36, LOX-1 expression.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R363

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