內(nèi)源性硫化氫對巨噬細胞吞噬脂質(zhì)過程中鈣化和BMP表達的影響
發(fā)布時間：2018-10-29 20:33
【摘要】：背景與目的:異位鈣化即非骨組織發(fā)生礦物質(zhì)沉積,主要是鈣鹽沉積,血管組織鈣化是其中的一種,分為中膜鈣化和內(nèi)膜鈣化。后者與動脈粥樣硬化密切相關(guān),早期參與的細胞主要是巨噬細胞和肥大細胞。硫化氫在心血管系統(tǒng)中具有重要的生理與病理作用,本實驗擬利用L-半胱氨酸作為硫化氫的內(nèi)源性供體,在β-甘油磷酸鈉(β-sodium glycerophosphate,β-GP)和氧化低密度脂蛋白(oxidized low density lipoprotein,oxLDL)誘導的巨噬細胞鈣鹽沉積的細胞模型上研究內(nèi)源性硫化氫對巨噬細胞吞噬脂質(zhì)過程中鈣化和骨形成蛋白(Bone morphogenetic protein,BMP)表達的影響及其機制,為進一步探討其對內(nèi)膜鈣化的作用提供實驗依據(jù)。 方法: 實驗一鈣化細胞模型的建立 小鼠單核巨噬細胞RAW264.7用5mmol/L的β-GP和40mg/mL的oxLDL孵育3天,von Kossa染色形態(tài)學觀察細胞鈣鹽沉積,原子吸收分光光度法測定細胞鈣含量。 實驗二不同濃度L-半胱氨酸對細胞鈣化和BMP表達的影響 1、正常對照組:含10%胎牛血清RPMI-1640培養(yǎng)基。 2、鈣化組:40mg/mL oxLDL和5mmol/L的β-GP培養(yǎng)基孵育細胞。 3、不同濃度L-半胱氨酸組(50,100,200mmol/L):鈣化組細胞培養(yǎng)基的基礎(chǔ)上加入相應(yīng)濃度L-半胱氨酸(50,100,200mmol/L),孵育細胞4天。 實驗三L-半胱氨酸處理不同時間對細胞鈣化和BMP表達的影響 1、正常對照組:含10%胎牛血清RPMI-1640培養(yǎng)基。 2、不同時間處理組(0,2,4,6d):最佳濃度L-半胱氨酸處理鈣化組細胞不同時間(0,2,4,6d)。 實驗四PPG對細胞鈣化和BMP表達的影響 1、正常對照組:10%胎牛血清RPMI-1640培養(yǎng)基。 2、不同濃度PPG處理鈣化細胞組(0,2,4,8mmol/L):不同濃度PPG孵育鈣化細胞組12h。 結(jié)果: 鈣化組細胞von Kossa染色見細胞內(nèi)有大量褐色顆粒聚集,鈣含量高于對照組2.1倍(P0.01), BMP mRNA和蛋白表達分別是正常對照組的1.6倍和1.8倍(P0.05),L-半胱氨酸組較鈣化組細胞,細胞von Kossa染色褐色顆粒減少,鈣含量降低,BMP蛋白和mRNA表達也呈下降趨勢,特別是50mmol/L的L-半胱氨酸組作用最顯著von Kossa染色幾乎未見褐色顆粒,其BMP mRNA和蛋白表達低于鈣化組1.6倍和2.2倍(P0.05),接近正常對照組。PPG組結(jié)果相反,即隨著PPG濃度的升高,細胞鈣化程度加重,von Kossa染色可見大量褐色顆粒,BMP mRNA和蛋白的表達也隨濃度升高而增加。 結(jié)論: 1、內(nèi)源性硫化氫可以減少巨噬細胞吞噬脂質(zhì)過程中的細胞內(nèi)鈣含量和鈣沉積; 2、內(nèi)源性硫化氫可以下調(diào)巨噬細胞吞噬脂質(zhì)過程中BMP的表達水平。
[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.
【學位授予單位】：南華大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R363
本文編號：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.
【學位授予單位】：南華大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R363
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