本文選題：硫化氫 + 炔丙基甘氨酸　； 參考：《蘇州大學》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胞內吞氧化型低密度脂蛋白的影響，并對其分子學機制進行初步探討，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天有細胞爬出，7～9天可進行第一次傳代。實驗選用3～4代VSMC。根據(jù)不同檢測方法可分為：①用于MTT檢測細胞活性的細胞接種于96孔板，按5×104/mL細胞密度每孔200μl；②用于油紅O染色、免疫細胞化學檢測的細胞接種于放有小圓玻片的24孔板內，按1×105/ml細胞密度每孔1ml；③.用于免疫印跡檢測的細胞接種于無菌6孔板中，按1×106/ml細胞密度，每孔2ml。 實驗分組： 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脂質含量與免疫細胞化學法檢測CD36、LOX-1受體表達：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：分為對照組、ox-LDL（80μg/ml）組、ox-LDL（80μg/ml）+NaHS（25、50、100、200μmol/L）組。 結果： 1.MTT結果顯示，NaHS濃度在200μmol/L以下時，對VSMC的活力沒有明顯影響，當NaHS濃度增加到500和1000μmol/L后細胞活力明顯下降。 2.通過油紅O染色觀察外源性H2S（以NaHS為供體）對VSMC脂質攝取的影響。結果發(fā)現(xiàn)，與對照組相比，VSMC與ox-LDL單獨孵育后胞內脂質顯著增多，當用CSE（胱硫醚-γ-裂解酶）抑制劑PPG作用后，脂質沉積更明顯，而H2S處理后的VSMC胞內脂質明顯減少。 3.用DiI-oxLDL處理VSMC，Confocal觀察VSMC對ox-LDL的內吞作用，主要觀察外加DiI-oxLDL在VSMC胞內的積聚。結果觀察到H2S能抑制VSMC對ox-LDL的內吞，而PPG則增強這種內吞作用，與油紅O染色結果相符。 4.免疫細胞化學法檢測VSMC的CD36、LOX-1受體表達，對H2S抑制VSMC內吞ox-LDL的分子學機制進行初步探討。結果顯示，ox-LDL能明顯誘導VSMC的CD36、LOX-1受體表達，H2S處理后可下調這兩種受體的表達，，PPG作用后則使CD36、LOX-1受體表達上調。 5.為進一步確定H2S抑制VSMC內吞脂質的機制是否與其下調CD36、LOX-1有關，本實驗還采用了Western blot方法分析NaHS對VSMC的CD36、LOX-1蛋白表達的影響。結果發(fā)現(xiàn)，ox-LDL顯著增強誘導VSMC的CD36、LOX-1蛋白表達，而H2S對兩種受體蛋白表達的抑制作用呈劑量依賴性。 結論: 1.外源性H2S可通過抑制VSMC對ox-LDL的內吞減少脂質在胞內的沉積。 2. H2S能夠抑制VSMC內脂質沉積，其機制與其下調CD36、LOX-1表達有關。
[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.
【學位授予單位】：蘇州大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R363

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