本文關(guān)鍵詞：硫化氫對巨噬細胞源性泡沫細胞形成和動脈粥樣硬化斑塊脂質(zhì)蓄積的影響　出處：《南華大學(xué)》2011年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 硫化氫 氧化低密度脂蛋白 單核源性巨噬細胞 硫化氫 泡沫細胞 氧化低密度脂蛋白 清道夫受體 �；o酶 A：膽固醇�；D(zhuǎn)移酶-1 動脈粥樣硬化 apoE-/-小鼠 泡沫細胞 脂質(zhì)蓄積 硫化氫

【摘要】：研究背景：內(nèi)源性硫化氫(hydrogen sulfide, H2S)可在許多哺乳動物組織、細胞中產(chǎn)生，其合成途徑主要為胱硫醚γ-裂解酶(cystathionine-γ-lyase,CSE)和胱硫醚-β合酶(cystathionine-synthase,CBS)催化L-半胱氨酸而生成。H2S具有廣泛的作用，，被認為是第三個氣體信號分子。冠心病人冠狀動脈動脈粥樣硬化(atherosclerosis,As)病變程度和病變血管數(shù)量與血漿H2S水平呈負相關(guān)，但機制有待闡明。單核源性巨噬細胞是促進As發(fā)生發(fā)展的一類重要的炎癥細胞。氧化低密度脂蛋白(oxidized low-densitylipoprotein, oxLDL)是As的獨立危險因素，可激活巨噬細胞并調(diào)節(jié)巨噬細胞功能。然而，人單核源性巨噬細胞是否存在H2S生成體系，oxLDL是否調(diào)節(jié)巨噬細胞H2S的生成，目前尚無報道。 目的：研究人單核源性巨噬細胞是否存在H2S生成體系以及oxLDL對巨噬細胞生成H2S的調(diào)節(jié)作用。 方法：采用亞甲基藍法結(jié)合分光光度儀測H2S的生成，RT-PCR和Western blot檢測CSE和CBS的mRNA和蛋白水平。 結(jié)果：人單核源性巨噬細胞有豐富的CSE和少量的CBSmRNA表達，并內(nèi)源性產(chǎn)生H2S。L-半胱氨酸（L-cysteine, CSE和CBS的作用底物，2mmol/L）增加巨噬細胞內(nèi)H2S產(chǎn)生和釋放，較對照組增加約28.1%(P 0.05)；而炔丙基甘氨酸(DL-propargylglycine，PPG： CSE抑制劑，3mmol/L）和羥胺（hydroxylamine， HA：CBS抑制劑，100μmol/L）抑制巨噬細胞內(nèi)H2S的產(chǎn)生和釋放，分別較對照組減少約26.8%和27.6%(P 0.05)。OxLDL（100μg/ml）降低巨噬細胞H2S合成酶活性約23.3%，呈時間和劑量依賴性減少細胞上清中H2S水平，并解除L-半胱氨酸對H2S生成的促進作用，但與PPG和HA協(xié)同抑制H2S產(chǎn)生。進一步研究發(fā)現(xiàn)，OxLDL明顯下調(diào)巨噬細胞CSE mRNA和蛋白水平，輕微下調(diào)CBS mRNA水平。結(jié)論：1.人單核源性巨噬細胞內(nèi)存在H2S生成系統(tǒng)，且以H2S/CSE為主，能生成內(nèi)源性H2S。 2. OxLDL抑制巨噬細胞內(nèi)源性H2S生成。 研究背景：研究顯示H2S對As發(fā)生的一些環(huán)節(jié)具有拮抗效應(yīng)，如抑制大鼠主動脈平滑肌細胞(smooth muscle cells,SMC)增殖，抑制動脈內(nèi)皮細胞粘附分子表達等。由于As形成是一個極其復(fù)雜的過程，因此有必要進一步探索H2S的潛在抗As效應(yīng)。巨噬細胞源性泡沫細胞在As發(fā)生發(fā)展中起至關(guān)重要的作用。H2S是否影響泡沫細胞形成，尚未有報道，值得探索。 目的：探索硫化氫對巨噬細胞源性泡沫細胞形成的影響及其機制。 方法：采用佛玻酯誘導(dǎo)單核細胞（人外周血原始單核細胞或THP-1細胞）分化為巨噬細胞。用油紅O染色和高效液相色譜分析巨噬細胞內(nèi)脂質(zhì)及總膽固醇(total cholesterol，TC)、膽固醇酯(cholesterol ester，CE)含量，熒光顯微鏡分析oxLDL結(jié)合和攝��；以Western blot檢測清道夫受體A (scavenger receptor A，SR-A)， CD36和�；o酶A:膽固醇�；D(zhuǎn)移酶-1(acyl-coenzyme A:cholesterol acyltransferase-1，ACAT-1)表達，ELISA法測細胞上清TNF-α水平。 結(jié)果：油紅O染色和高效液相色譜結(jié)果顯示，巨噬細胞與oxLDL單獨孵育可明顯增加細胞內(nèi)中性脂質(zhì)及細胞內(nèi)TC、CE含量。硫氫化鈉(sodium hydrosulfide，NaHS：H2S供體)顯著減輕oxLDL誘導(dǎo)的細胞內(nèi)脂質(zhì)蓄積，減少TC、CE含量和CE/TC比值。而PPG（H2S生成酶CSE抑制劑）促進細胞內(nèi)脂質(zhì)蓄積，增加TC、CE含量和CE/TC比值。巨噬細胞孵育DiI-oxLDL后大量結(jié)合并攝取DiI-oxLDL，NaHS抑制巨噬細胞結(jié)合和攝取DiI-oxLDL， PPG促進巨噬細胞結(jié)合和攝取DiI-oxLDL。進一步研究發(fā)現(xiàn)，oxLDL可顯著誘導(dǎo)巨噬細胞CD36、SR-A和ACAT-1表達，而NaHS明顯抑制oxLDL誘導(dǎo)的CD36、SR-A和ACAT-1表達。KATP通道阻滯劑格列苯脲阻止、ERK1/2抑制劑PD98059促進NaHS的上述效應(yīng)。NaHS也可減少TNF-α的產(chǎn)生和分泌，并部分抑制TNF-α誘導(dǎo)的CD36表達。 結(jié)論： H2S可能部分經(jīng)KATP/ERK1/2環(huán)節(jié)或部分經(jīng)抑制TNF-α分泌下調(diào)巨噬細胞CD36或SR-A、ACAT-1表達，減少oxLDL結(jié)合和攝取，抑制巨噬細胞源性泡沫細胞形成。 研究背景：文獻報道哺乳動物的動脈組織(主動脈、冠狀動脈、小動脈)都含有H2S生成酶CSE，能合成和產(chǎn)生H2S，提示H2S可能參與血管功能的調(diào)節(jié)。本課題的前期研究顯示H2S可抑制巨噬細胞內(nèi)脂質(zhì)蓄積，提示這一氣體信號分子可能減少As斑塊脂質(zhì)含量，抑制粥樣斑塊形成。 目的：研究H2S對As斑塊脂質(zhì)蓄積的影響。 方法：8周齡雄性apoE-/-小鼠，飼喂高脂高膽固醇飲食，分別腹腔注射溶媒（生理鹽水）、NaHS、PPG。8周齡雄性C57BL/6J小鼠用作空白對照。20周后，取材分析。采用油紅O和蘇丹IV染色檢測主動脈根部病變及主動脈內(nèi)膜面脂質(zhì)含量。采用HE、Movat5’套染檢測斑塊泡沫細胞。以免疫組織化學(xué)法或免疫熒光法測CSE、SR-A、CD36和ACAT-1抗原水平及巨噬細胞含量，Real-time PCR測SR-A、CD36及ACAT-1mRNA水平。用ELISA法測血清TNF-α水平。亞甲基藍法結(jié)合分光光度儀測血漿H2S濃度和主動脈H2S產(chǎn)率。以酶法檢測血脂水平。 結(jié)果：各組小鼠的血脂水平和體重?zé)o明顯不同。ApoE-/-主動脈根部病變有CSE表達。與C57BL/6小鼠比較，apoE-/-生理鹽水組小鼠的血漿H2S水平減少。而與生理鹽水組比較，NaHS提高apoE-/-小鼠血漿H2S水平約21.4%（P㩳0.05），PPG不僅使主動脈H2S合成活性降低約35.6%（P㩳0.05），還使血漿H2S水平降低約22.4%（P㩳0.05）。油紅O和蘇丹IV染色結(jié)果顯示，NaHS減少主動脈根部病變脂質(zhì)陽性區(qū)百分比和主動脈內(nèi)膜面脂質(zhì)陽性區(qū)面積，分別較生理鹽水組減少23.2%(P㩳0.05)和49.0%(P㩳0.05)。而與生理鹽水組比較，PPG組主動脈根部病變脂質(zhì)陽性區(qū)百分比增加31.3%(P㩳0.01)，主動脈內(nèi)膜面脂質(zhì)陽性區(qū)面積擴大75.4%(P㩳0.01)。與脂質(zhì)陽性區(qū)面積變化一致的是，NaHS減少主動脈根部病變總面積(P㩳0.05)，而PPG增加主動脈根部病變總面積(P㩳0.05)。Movat’5和HE染色結(jié)果顯示，NaHS較生理鹽水顯著減少主動脈根部泡沫細胞累積和泡沫細胞面積；而PPG明顯增加泡沫細胞累積和泡沫細胞面積。與Movat’5結(jié)果相關(guān)的是，NaHS抑制主動脈根部病變巨噬細胞聚積、減少SR-A、CD36和ACAT-1表達；而PPG促進主動脈病變巨噬細胞蓄積和增加SR-A、CD36、ACAT-1表達。NaHS也明顯降低血清TNF-α水平，而PPG增加血清TNF-α水平。 結(jié)論：H2S可抑制apoE-/-小鼠As斑塊脂質(zhì)蓄積，其機制可能與減少斑塊內(nèi)巨噬細胞含量、下調(diào)SR-A、CD36、ACAT-1表達、降低血清TNF-α水平有關(guān)。
[Abstract]:Background: endogenous hydrogen sulfide (hydrogen sulfide, H2S) in many mammalian tissues, cells, the synthesis pathway for cystathionine gamma lyase (cystathionine- R -lyase, CSE) and cystathionine beta synthase (cystathionine-synthase, CBS) L- generated.H2S catalytic cysteine has many functions. Third is considered to be a gaseous signal molecule. Coronary heart disease coronary artery atherosclerosis (atherosclerosis, As) and the severity of lesion number was negatively correlated with the level of H2S, but the mechanism remains to be elucidated. Monocyte derived macrophages are an important class of inflammatory cells and promote the occurrence and development of As. The oxidation of low density lipoprotein (oxidized Low-densitylipoprotein, oxLDL) is an independent risk factor of As, can activate macrophages and macrophage function. However, whether the human monocyte derived macrophages H2S generation system o It is not yet reported whether xLDL regulates the formation of macrophage H2S.
Objective: To investigate whether there is a H2S generation system in human mononuclear macrophages and the regulation of oxLDL on the formation of H2S in macrophages.
Methods: the formation of H2S was measured by methylene blue and spectrophotometer, and the levels of mRNA and protein of CSE and CBS were detected by RT-PCR and Western blot.
Results: human monocyte derived macrophages have a rich CSE and a small amount of expression of CBSmRNA and H2S.L- cysteine (L-cysteine, endogenous substrates, CSE and CBS 2mmol/L) increased macrophage H2S production and release, compared with the control group increased by about 28.1% (P 0.05); and propargylglycine (DL-propargylglycine, PPG:CSE inhibitor. 3mmol/L (hydroxylamine, HA:CBS) and hydroxylamine inhibitor, 100 mol/L) inhibited the production and release of H2S in macrophages, about 26.8% and 27.6% respectively compared with the control group decreased (P 0.05).OxLDL (100 g/ml) reduced H2S synthase activity and low macrophage of about 23.3%, in a time and dose-dependent decrease of H2S level in the culture supernatant. And the release of L- cysteine on the formation of H2S role, but with the PPG and HA synergistic inhibition of H2S. Further studies showed that OxLDL significantly reduced macrophage CSE and protein levels of mRNA, CBS slightly reduced MRNA level. Conclusion: there is a H2S generation system in 1. human mononuclear macrophages, which is based on H2S/CSE and can produce endogenous H2S..
2. OxLDL inhibited the endogenous H2S formation of macrophages.
Background: studies show that H2S has an antagonistic effect on some aspects of As, such as inhibition of rat aortic smooth muscle cells (smooth muscle cells, SMC) proliferation, inhibit the expression of adhesion molecules in endothelial cells. The As formation is a very complicated process, so there is potential for further exploration of the anti As effect of H2S. Macrophage derived foam cells in play crucial role in the.H2S affect the formation of foam cells in the occurrence and development of As have not been reported, is worth exploring.
Objective: To explore the effect of hydrogen sulfide on the formation of macrophage derived foam cells and its mechanism.
Methods: the Buddha ester induced monocyte (glassy original human peripheral blood mononuclear cells or THP-1 cells) differentiated into macrophages. Macrophages and lipid analysis of total cholesterol in oil red O staining and high performance liquid chromatography (total cholesterol, TC (cholesterol), cholesterol ester ester, CE) content, oxLDL binding and analysis the uptake of fluorescence microscope; Western blot A (scavenger receptor detection of scavenger receptor A, CD36 and SR-A), acyl coenzyme A: cholesterol acyltransferase (acyl-coenzyme -1 A:cholesterol Acyltransferase-1, ACAT-1) expression measured cell supernatant levels of TNF- alpha ELISA method.
Results: the results of oil red O staining and HPLC showed that macrophages incubated with oxLDL alone significantly increased the neutral lipid and intracellular TC, CE. The content of sodium hydrosulfide (sodium hydrosulfide, NaHS:H2S donor) significantly reduced the oxLDL induced lipid accumulation in cells, decrease TC, CE content and CE/TC ratio. PPG (H2S CSE synthase inhibitor) to promote lipid accumulation, increase TC, CE content and CE/TC ratio. Macrophages after DiI-oxLDL incubation and combining with a large number of intake of DiI-oxLDL, NaHS inhibited macrophage binding and uptake of DiI-oxLDL, PPG promote macrophage binding and uptake DiI-oxLDL. further study found that oxLDL can significantly induce the expression of SR-A and CD36 in macrophages. ACAT-1 and NaHS significantly inhibited oxLDL induced CD36, SR-A and ACAT-1 expression of.KATP channel blocker glibenclamide block, ERK1/2 inhibitor PD98059 on.N the effect of NaHS AHS also reduces the production and secretion of TNF- alpha, and partially inhibits the expression of CD36 induced by TNF- alpha.
Conclusion: H2S may partly inhibit TNF- alpha secretion by KATP/ERK1/2 or part, down regulate macrophage CD36 or SR-A and ACAT-1 expression, reduce oxLDL binding and uptake, and inhibit macrophage derived foam cell formation.
Background: arterial tissues reported in mammals (aorta, coronary artery, arteriole) contains H2S synthase CSE, synthesis and production of H2S, suggesting that H2S may participate in the regulation of vascular function. Our previous study showed that H2S can inhibit lipid accumulation in macrophages, suggesting a gaseous signal molecule may reduce As lipid plaque the content of inhibition of plaque formation.
Objective: To study the effect of H2S on the accumulation of lipid in As plaque.
Methods: 8 week old male apoE-/- mice were fed with high fat and high cholesterol diet were injected solvent (saline), NaHS, PPG.8 week old male C57BL/6J mice were used as blank control. After.20 weeks, specimens were harvested and analyzed by oil red O and Sultan IV staining of aortic root lesions and aortic intima lipid content by HE. Movat5 ', or detect plaque foam cells by immunohistochemical method and immunofluorescence method to measure CSE, SR-A, CD36 and ACAT-1 antigen levels and macrophage content, Real-time PCR SR-A, CD36 and ACAT-1mRNA level. Serum TNF- levels were measured by ELISA method. The methylene blue method combined with the measurement of plasma H2S concentration and aortic H2S yield points light photometer. To detect the level of lipids enzyme method.
Results: the mice serum lipid levels and body weight not significantly different.ApoE-/- aortic root disease has the expression of CSE. Compared with C57BL/6 mice, the levels of plasma H2S apoE-/- normal saline group were reduced. Compared with the normal saline group, NaHS increased the levels of H2S apoE-/- in plasma of about 21.4% (P? 0.05), PPG not only makes the aortic H2S synthesis activity was decreased by 35.6% (P? 0.05), the plasma level of H2S decreased by about 22.4% (P? 0.05). Oil red O and Sultan IV staining showed that NaHS positive area decreased aortic root disease and aortic intimal lipid percentage of positive area lipid area, respectively, compared with the saline group decreased 23.2% (P? 0.05) and 49% (P? 0.05). Compared with the normal saline group, PPG group of aortic root lesions was 31.3% percentage increase in lipid (P? 0.01), aortic intimal area was enlarged by 75.4% surface lipid (P? 0.01). The area change and positive area of lipid The consensus is that NaHS reduced the total area of the aortic root lesions (P? 0.05), but PPG increased aortic root disease total area (P? 0.05).Movat 5 and HE staining showed that NaHS was significantly reduced compared with the saline area of the aortic root cells and foam foam cell accumulation; while PPG significantly increased the area of cells and foam cell accumulation. And Movat '5 results are related to the inhibition of NaHS, aortic root disease reduced SR-A, macrophage accumulation, the expression of CD36 and ACAT-1; and PPG promote macrophage accumulation and increased aortic SR-A, CD36, ACAT-1 expression of.NaHS also decreased serum TNF- levels, while PPG increased serum TNF- levels.
Conclusion: H2S can inhibit the accumulation of As plaques in apoE-/- mice, and its mechanism may be related to the reduction of macrophage content in plaques, down regulation of SR-A, CD36, ACAT-1 expression and decrease of serum TNF- alpha level.

【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2011
【分類號】：R363

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