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
发布时间：2018-10-29 20:33
【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(P0.01), BMP mRNA和蛋白表达分别是正常对照组的1.6倍和1.8倍(P0.05),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(P0.05),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。
[Abstract]:Background & AIM: ectopic calcification is the mineral deposition of non-bone tissues, mainly calcium salt deposition. Vascular calcification is one of them, which is divided into medial calcification and intimal calcification. The latter is closely related to atherosclerosis, and the cells involved in the early stage are mainly macrophages and mast cells. Hydrogen sulfide plays an important physiological and pathological role in the cardiovascular system. In this study, L- cysteine was used as an endogenous donor of hydrogen sulfide, and 尾-sodium glycerophosphate, was used in this study. Calcium deposition of macrophages induced by 尾-GP) and oxidized low density lipoprotein (oxidized low density lipoprotein,oxLDL The effect of BMP) and its mechanism provide experimental basis for further study of its effect on intimal calcification. Methods: the mouse macrophage RAW264.7 was incubated with 尾-GP of 5mmol/L and oxLDL of 40mg/mL for 3 days with, von Kossa staining to observe the calcium deposition of mouse macrophages. The content of calcium in cells was determined by atomic absorption spectrophotometry. Experiment 2 effects of different concentrations of L-cysteine on calcification and BMP expression. 1 normal control group: RPMI-1640 medium containing 10% fetal bovine serum. 2, calcification group: 40mg/mL oxLDL and 5mmol/L were incubated on 尾-GP medium. 3. Different concentrations of L-cysteine (50100200mmol/L): calcified cells were incubated with L- cysteine (50100200mmol/L) for 4 days. The effects of L- cysteine treatment on calcification and BMP expression at different time 1. Normal control group: RPMI-1640 medium containing 10% fetal bovine serum. 2, different time treatment group (0o 2n 4U 6 d): the best concentration of L- cysteine treated calcification group cells at different time (0T 2n 4U 6 d). Effect of PPG on calcification and BMP expression. 1 normal control group: 10% fetal bovine serum RPMI-1640 medium. 2. The calcified cells were treated with different concentrations of PPG for 12 h (0 ~ (2) O ~ (2 +) and 8 mmol 路L ~ (-1) 路L ~ (-1) PPG. Results: the von Kossa staining of calcified cells showed that a large number of brown granules accumulated in the cells, and the calcium content was 2.1-fold higher than that in the control group (P0.01). The expression of BMP mRNA and protein were 1.6-fold and 1.8-fold of normal control group respectively (P0.05). Compared with calcified cells, von Kossa staining brown granules decreased and calcium content decreased in L- cysteine group. The expression of BMP protein and mRNA also decreased, especially in the L- cysteine group of 50mmol/L. The expression of BMP mRNA and protein in 50mmol/L group was 1.6-fold and 2.2-fold lower than that in calcified group (P0.05). The results of PPG group were opposite, that is, with the increase of PPG concentration, the degree of cell calcification increased and, von Kossa staining showed that a large number of brown granules, BMP mRNA and protein expression also increased with the increase of concentration. Conclusion: 1. Endogenous hydrogen sulfide can reduce intracellular calcium content and calcium deposition during phagocytosis of macrophages, and endogenous hydrogen sulfide can down-regulate the expression of BMP during macrophage phagocytosis.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363
本文编号：2298738
[Abstract]:Background & AIM: ectopic calcification is the mineral deposition of non-bone tissues, mainly calcium salt deposition. Vascular calcification is one of them, which is divided into medial calcification and intimal calcification. The latter is closely related to atherosclerosis, and the cells involved in the early stage are mainly macrophages and mast cells. Hydrogen sulfide plays an important physiological and pathological role in the cardiovascular system. In this study, L- cysteine was used as an endogenous donor of hydrogen sulfide, and 尾-sodium glycerophosphate, was used in this study. Calcium deposition of macrophages induced by 尾-GP) and oxidized low density lipoprotein (oxidized low density lipoprotein,oxLDL The effect of BMP) and its mechanism provide experimental basis for further study of its effect on intimal calcification. Methods: the mouse macrophage RAW264.7 was incubated with 尾-GP of 5mmol/L and oxLDL of 40mg/mL for 3 days with, von Kossa staining to observe the calcium deposition of mouse macrophages. The content of calcium in cells was determined by atomic absorption spectrophotometry. Experiment 2 effects of different concentrations of L-cysteine on calcification and BMP expression. 1 normal control group: RPMI-1640 medium containing 10% fetal bovine serum. 2, calcification group: 40mg/mL oxLDL and 5mmol/L were incubated on 尾-GP medium. 3. Different concentrations of L-cysteine (50100200mmol/L): calcified cells were incubated with L- cysteine (50100200mmol/L) for 4 days. The effects of L- cysteine treatment on calcification and BMP expression at different time 1. Normal control group: RPMI-1640 medium containing 10% fetal bovine serum. 2, different time treatment group (0o 2n 4U 6 d): the best concentration of L- cysteine treated calcification group cells at different time (0T 2n 4U 6 d). Effect of PPG on calcification and BMP expression. 1 normal control group: 10% fetal bovine serum RPMI-1640 medium. 2. The calcified cells were treated with different concentrations of PPG for 12 h (0 ~ (2) O ~ (2 +) and 8 mmol 路L ~ (-1) 路L ~ (-1) PPG. Results: the von Kossa staining of calcified cells showed that a large number of brown granules accumulated in the cells, and the calcium content was 2.1-fold higher than that in the control group (P0.01). The expression of BMP mRNA and protein were 1.6-fold and 1.8-fold of normal control group respectively (P0.05). Compared with calcified cells, von Kossa staining brown granules decreased and calcium content decreased in L- cysteine group. The expression of BMP protein and mRNA also decreased, especially in the L- cysteine group of 50mmol/L. The expression of BMP mRNA and protein in 50mmol/L group was 1.6-fold and 2.2-fold lower than that in calcified group (P0.05). The results of PPG group were opposite, that is, with the increase of PPG concentration, the degree of cell calcification increased and, von Kossa staining showed that a large number of brown granules, BMP mRNA and protein expression also increased with the increase of concentration. Conclusion: 1. Endogenous hydrogen sulfide can reduce intracellular calcium content and calcium deposition during phagocytosis of macrophages, and endogenous hydrogen sulfide can down-regulate the expression of BMP during macrophage phagocytosis.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363
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