内源性硫化氢对巨噬细胞吞噬脂质过程中钙化和BMP表达的影响
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摘要
背景与目的:异位钙化即非骨组织发生矿物质沉积,主要是钙盐沉积,血管组织钙化是其中的一种,分为中膜钙化和内膜钙化。后者与动脉粥样硬化密切相关,早期参与的细胞主要是巨噬细胞和肥大细胞。硫化氢在心血管系统中具有重要的生理与病理作用,本实验拟利用L-半胱氨酸作为硫化氢的内源性供体,在β-甘油磷酸钠(β-sodium glycerophosphate,β-GP)和氧化低密度脂蛋白(oxidized low density lipoprotein,oxLDL)诱导的巨噬细胞钙盐沉积的细胞模型上研究内源性硫化氢对巨噬细胞吞噬脂质过程中钙化和骨形成蛋白(Bone morphogenetic protein,BMP)表达的影响及其机制,为进一步探讨其对内膜钙化的作用提供实验依据。
方法:
实验一钙化细胞模型的建立
小鼠单核巨噬细胞RAW264.7用5mmol/L的β-GP和40mg/mL的oxLDL孵育3天,von Kossa染色形态学观察细胞钙盐沉积,原子吸收分光光度法测定细胞钙含量。
实验二不同浓度L-半胱氨酸对细胞钙化和BMP表达的影响
1、正常对照组:含10%胎牛血清RPMI-1640培养基。
2、钙化组:40mg/mL oxLDL和5mmol/L的β-GP培养基孵育细胞。
3、不同浓度L-半胱氨酸组(50,100,200mmol/L):钙化组细胞培养基的基础上加入相应浓度L-半胱氨酸(50,100,200mmol/L),孵育细胞4天。
实验三L-半胱氨酸处理不同时间对细胞钙化和BMP表达的影响
1、正常对照组:含10%胎牛血清RPMI-1640培养基。
2、不同时间处理组(0,2,4,6d):最佳浓度L-半胱氨酸处理钙化组细胞不同时间(0,2,4,6d)。
实验四PPG对细胞钙化和BMP表达的影响
1、正常对照组:10%胎牛血清RPMI-1640培养基。
2、不同浓度PPG处理钙化细胞组(0,2,4,8mmol/L):不同浓度PPG孵育钙化细胞组12h。
结果:
钙化组细胞von Kossa染色见细胞内有大量褐色颗粒聚集,钙含量高于对照组2.1倍(P<0.01), BMP mRNA和蛋白表达分别是正常对照组的1.6倍和1.8倍(P<0.05),L-半胱氨酸组较钙化组细胞,细胞von Kossa染色褐色颗粒减少,钙含量降低,BMP蛋白和mRNA表达也呈下降趋势,特别是50mmol/L的L-半胱氨酸组作用最显著von Kossa染色几乎未见褐色颗粒,其BMP mRNA和蛋白表达低于钙化组1.6倍和2.2倍(P<0.05),接近正常对照组。PPG组结果相反,即随着PPG浓度的升高,细胞钙化程度加重,von Kossa染色可见大量褐色颗粒,BMP mRNA和蛋白的表达也随浓度升高而增加。
结论:
1、内源性硫化氢可以减少巨噬细胞吞噬脂质过程中的细胞内钙含量和钙沉积;
2、内源性硫化氢可以下调巨噬细胞吞噬脂质过程中BMP的表达水平。
Objective:
Heterotopic calcification, namely, the mineral deposition occurred in the nonosteonal tissue, mainly refers to the deposition of calcium salt, and one kind, the calcification of vascular tissue, is classified into tunica medial calcification, and tunica intimal calcification. The tunica intimal calcification is closely relevant to artherosclerosis, and its early involvements primarily include macrophages and clasmatoblast. The solfurated hydrogen possesses important physiological action and pathological action of cardiovascular. With L-cysteine as the endogenous donor of solfurated hydrogen, based on the model of calcium salt deposition in macrophages which is guided byβ-GP and oxLDL, this research attempts to study the effects of endogenous sulfureted hydrogen on calcification during macrophages lipid loading and the factors behind them, providing some experimental evidence for further exploration of its effects on the tunica intimal calcification.
Methods:
Expriment 1: Establish the model of the calcification: Incubate RAW264.7 with 40mg/ml oxLDL and 5mmol/Lβ-GP for three days; obseve calcium deposition by von Kossa staining and determine cumulation content by atomic absorption spectrophotometry.
Expriment 2: Effect on the cell calcification of endogenous sulfureted hydrogen with different concentration:
1.Control group: cell incubated in RPMI -1640 with 10% tetal caif serum .
2.Calfication and 0mmol/L L-lysine groups:cell incubated with oxLDL,β-GP and 0mmol/L L-lysine.
3. Calfication and 50mmol/L L-lysine groups:cell incubarted with oxLDL,β-GP and 50mmol/L L-lysine; calfication and 100mmol/L L-lysine groups:cell incubarted with oxLDL,β-GP and 100mmol/L L-lysine; calfication and 200mmol/L L-lysine groups:cell incubarted with oxLDL,β-GP and 200mmol/L L-lysine.
Expriment 3: Effect of endogenous sulfureted hydrogen on the cell calcification in different periods of time:
1. Control group: cell incubated in RPMI -1640 with 10% tetal calf serum.
2. 0d :cell incubated with oxLDL,β-GP and bestL-lysine contration for 0 day; 2d: cell incubated with cell were incubated with oxLDL,β-GP and best L-lysine contration for 2 days; 4d: cell incubated with oxLDL,β-GP and best L-lysine contration for 4 days; 6d: cell incubated with oxLDL,β-GP and best L-lysine contration for 6 days.
Expriment 4: Effects of PPG on cell calcification:
RAW264.7 was incubated with oxLD,β-GP and PPG at different concentrations(0,2,4,8mmol/L) for 4 days.then we detected calcium content, calcium deposition and OPN mRNA and protein eapression were determined by RT-PCR and Westen blot, respectively. Raw264.7 were divided into five groups as followings:1) control group:cell incubated in RPMI -1640 with 10% tetal caif serum . 2) calfication and 0mmol/L PPG group:cell incubarted with oxLDL,β-GP and 0mmol/L PPG. 3) calfication and 2 mmol/L PPG group:cell incubarted with oxLDL,β-GP and 2mmol/L PPG. 4) calfication and 4mmol/L PPG group:cell incubated with oxLDL,β-GP and5 mmol/LPPG. 5) calfication and 8mmol/L PPG group:cell incubarted with oxLDL,β-GP and 8mmol/L PPG.
Results:
Compared with the control group, macrophages incubated with oxLDl andβ-glicerophosplale evidently showed a numbers of black particles, oxLDl andβ-glicerophosplale caused recrease the content of calium by 210%, and they also can recrease BMP mRNA and protein expression by 180% and 160% respectively. Compared with the calcification group, calcification macrophages incubated with L-cysteine macrophages showed less black particles,and also the contend of the calium. BMP mRNA and protein expression decreased too.expecially at the 50mmol/L level. L-cysteine caused a deceased BMP mRNA and protein expression by 161% and 220% respectively compared with calcification cell. Calcification cell incubated with PPG showed the opposite results compared with the cell incubated with L-cysteine..
Conclusion:
1.Endogenous sulfureted hydrogen can decrease the calcium content and calcium deposition during the macrophages lipid loading.
2.Endogenous sulfureted hydrogen can down-regulate BMP expression during the macrophages lipid loading.
方法:
实验一钙化细胞模型的建立
小鼠单核巨噬细胞RAW264.7用5mmol/L的β-GP和40mg/mL的oxLDL孵育3天,von Kossa染色形态学观察细胞钙盐沉积,原子吸收分光光度法测定细胞钙含量。
实验二不同浓度L-半胱氨酸对细胞钙化和BMP表达的影响
1、正常对照组:含10%胎牛血清RPMI-1640培养基。
2、钙化组:40mg/mL oxLDL和5mmol/L的β-GP培养基孵育细胞。
3、不同浓度L-半胱氨酸组(50,100,200mmol/L):钙化组细胞培养基的基础上加入相应浓度L-半胱氨酸(50,100,200mmol/L),孵育细胞4天。
实验三L-半胱氨酸处理不同时间对细胞钙化和BMP表达的影响
1、正常对照组:含10%胎牛血清RPMI-1640培养基。
2、不同时间处理组(0,2,4,6d):最佳浓度L-半胱氨酸处理钙化组细胞不同时间(0,2,4,6d)。
实验四PPG对细胞钙化和BMP表达的影响
1、正常对照组:10%胎牛血清RPMI-1640培养基。
2、不同浓度PPG处理钙化细胞组(0,2,4,8mmol/L):不同浓度PPG孵育钙化细胞组12h。
结果:
钙化组细胞von Kossa染色见细胞内有大量褐色颗粒聚集,钙含量高于对照组2.1倍(P<0.01), BMP mRNA和蛋白表达分别是正常对照组的1.6倍和1.8倍(P<0.05),L-半胱氨酸组较钙化组细胞,细胞von Kossa染色褐色颗粒减少,钙含量降低,BMP蛋白和mRNA表达也呈下降趋势,特别是50mmol/L的L-半胱氨酸组作用最显著von Kossa染色几乎未见褐色颗粒,其BMP mRNA和蛋白表达低于钙化组1.6倍和2.2倍(P<0.05),接近正常对照组。PPG组结果相反,即随着PPG浓度的升高,细胞钙化程度加重,von Kossa染色可见大量褐色颗粒,BMP mRNA和蛋白的表达也随浓度升高而增加。
结论:
1、内源性硫化氢可以减少巨噬细胞吞噬脂质过程中的细胞内钙含量和钙沉积;
2、内源性硫化氢可以下调巨噬细胞吞噬脂质过程中BMP的表达水平。
Objective:
Heterotopic calcification, namely, the mineral deposition occurred in the nonosteonal tissue, mainly refers to the deposition of calcium salt, and one kind, the calcification of vascular tissue, is classified into tunica medial calcification, and tunica intimal calcification. The tunica intimal calcification is closely relevant to artherosclerosis, and its early involvements primarily include macrophages and clasmatoblast. The solfurated hydrogen possesses important physiological action and pathological action of cardiovascular. With L-cysteine as the endogenous donor of solfurated hydrogen, based on the model of calcium salt deposition in macrophages which is guided byβ-GP and oxLDL, this research attempts to study the effects of endogenous sulfureted hydrogen on calcification during macrophages lipid loading and the factors behind them, providing some experimental evidence for further exploration of its effects on the tunica intimal calcification.
Methods:
Expriment 1: Establish the model of the calcification: Incubate RAW264.7 with 40mg/ml oxLDL and 5mmol/Lβ-GP for three days; obseve calcium deposition by von Kossa staining and determine cumulation content by atomic absorption spectrophotometry.
Expriment 2: Effect on the cell calcification of endogenous sulfureted hydrogen with different concentration:
1.Control group: cell incubated in RPMI -1640 with 10% tetal caif serum .
2.Calfication and 0mmol/L L-lysine groups:cell incubated with oxLDL,β-GP and 0mmol/L L-lysine.
3. Calfication and 50mmol/L L-lysine groups:cell incubarted with oxLDL,β-GP and 50mmol/L L-lysine; calfication and 100mmol/L L-lysine groups:cell incubarted with oxLDL,β-GP and 100mmol/L L-lysine; calfication and 200mmol/L L-lysine groups:cell incubarted with oxLDL,β-GP and 200mmol/L L-lysine.
Expriment 3: Effect of endogenous sulfureted hydrogen on the cell calcification in different periods of time:
1. Control group: cell incubated in RPMI -1640 with 10% tetal calf serum.
2. 0d :cell incubated with oxLDL,β-GP and bestL-lysine contration for 0 day; 2d: cell incubated with cell were incubated with oxLDL,β-GP and best L-lysine contration for 2 days; 4d: cell incubated with oxLDL,β-GP and best L-lysine contration for 4 days; 6d: cell incubated with oxLDL,β-GP and best L-lysine contration for 6 days.
Expriment 4: Effects of PPG on cell calcification:
RAW264.7 was incubated with oxLD,β-GP and PPG at different concentrations(0,2,4,8mmol/L) for 4 days.then we detected calcium content, calcium deposition and OPN mRNA and protein eapression were determined by RT-PCR and Westen blot, respectively. Raw264.7 were divided into five groups as followings:1) control group:cell incubated in RPMI -1640 with 10% tetal caif serum . 2) calfication and 0mmol/L PPG group:cell incubarted with oxLDL,β-GP and 0mmol/L PPG. 3) calfication and 2 mmol/L PPG group:cell incubarted with oxLDL,β-GP and 2mmol/L PPG. 4) calfication and 4mmol/L PPG group:cell incubated with oxLDL,β-GP and5 mmol/LPPG. 5) calfication and 8mmol/L PPG group:cell incubarted with oxLDL,β-GP and 8mmol/L PPG.
Results:
Compared with the control group, macrophages incubated with oxLDl andβ-glicerophosplale evidently showed a numbers of black particles, oxLDl andβ-glicerophosplale caused recrease the content of calium by 210%, and they also can recrease BMP mRNA and protein expression by 180% and 160% respectively. Compared with the calcification group, calcification macrophages incubated with L-cysteine macrophages showed less black particles,and also the contend of the calium. BMP mRNA and protein expression decreased too.expecially at the 50mmol/L level. L-cysteine caused a deceased BMP mRNA and protein expression by 161% and 220% respectively compared with calcification cell. Calcification cell incubated with PPG showed the opposite results compared with the cell incubated with L-cysteine..
Conclusion:
1.Endogenous sulfureted hydrogen can decrease the calcium content and calcium deposition during the macrophages lipid loading.
2.Endogenous sulfureted hydrogen can down-regulate BMP expression during the macrophages lipid loading.
引文
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[18]Bear M et al. Oxidized low-density lipoprotein acts synergistically with beta-glycerophosphate to induce osteoblast differentiation in primary cultures of vascular smooth muscle cells[J]. J Cell Biochem,2008,1097-4644.
[19]Chen NX et al. High glucose increases the expression of Cbfal and BMP-2 and enhances the calcification of vascular smooth muscle cells[J]. Nephrology Dialysis Transplantion, 2006, 21(12): 3435-3442.
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[22]Montecucco F et al. The immune response is involved in atherosclerotic plaque calcification: could the RANKL/RANK/OPG system be a marker of plaque instability?[J] Clin Immunol. 2007, 2007: 75805.
[23]Hsu H et al. Induction of calcification by serum depletion in cell culture: a model for focal calcification in aortas related to atherosclerosis[J]. Lipids Health Dis, 2008, 7: 2.
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[25]Schmermund A et al, The latest on the calcium story[J]. Am J Cardiol. 2002, 90(10C): 12-14.