AIM: To investigate the possibility of recombinant highdensity lipoprotein (rHDL) being a carrier for delivering antitumoral drug to hepatoma cells. METHODS: Recombinant complex of HDL and aclacinomycin (rHDL-ACM) was...AIM: To investigate the possibility of recombinant highdensity lipoprotein (rHDL) being a carrier for delivering antitumoral drug to hepatoma cells. METHODS: Recombinant complex of HDL and aclacinomycin (rHDL-ACM) was prepared by cosonication of apoproteins from HDL (Apo HDL) and ACM as well as phosphatidylcholine. Characteristics of the rHDL-ACM were elucidated by electrophoretic mobility, including the size of particles, morphology and entrapment efficiency. Binding activity of rHDL-ACM to human hepatoma cells was determined by competition assay in the presence of excess native HDL. The cytotoxicity of rHDL-ACM was assessed by MTT method. RESULTS: The density range of rHDL-ACM was 1.063-1.210 g/mL, and the same as that of native HDL. The purity of all rHDL-ACM preparations was more than 92%. Encapsulated efficiencies of rHDL-ACM were more than 90%. rHDL-ACM particles were typical sphere model of lipoproteins and heterogeneous in particle size. The average diameter was 31.26±5.62 nm by measure of 110 rHDL-ACM particles in the range of diameter of lipoproteins. rHDL-ACM could bind on SMMC-7721 cells, and such binding could be competed against in the presence of excess native HDL. rHDL-ACM had same binding capacity as native HDL. The cellular uptake of rHDL-ACM by SMMC-7721 hepatoma cells was significantly higher than that of free ACM at the concentration range of 0.5-10 μg/mL (P<0.01). Cytotoxicity of rHDL-ACM to SMMC-7721 cells was significantly higher than that of free ACM at concentration range of less than 5 ug/mL (P<0.01) and IC50 of rHDL-ACM was lower than IC50 of free ACM (1.68 nmol/L vs3 nmol/L). Compared to L02 hepatocytes, a normal liver cell line, the cellular uptake of rHDL-ACM by SMMC-7721 cells was significantly higher (P<0.01) and in a dose-dependent manner at the concentration range of 0.5-10 μg/mL.Cytotoxicity of the rHDL-ACM to SMMC- 7721 cells was significantly higher than that to L02 cells at concentration range of 1-7.5μg/mL (P<0.01). IC50 for SMMC-7721 cells (1.68 nmol/L) was lower than that for L02 cells (5.68 nmol/L), showing a preferential cytotoxicity of rHDL-ACM for SMMC-7721 cells. CONCLUSION: rHDL-ACM complex keeps the basic physical and biological binding properties of native HDL and shows a preferential cytotoxicity for SMMC-7721 hepatoma to normal L02 hepatocytes, HDL is a potential carrier for delivering lipophilic antitumoral drug to hepatoma cells.展开更多
Background In type 2 diabetes mellitus （T2DM）, high-density lipoprotein （HDL） impairs its anti-atherogenic properties and even develops to a pro-inflammatory and pro-atherogenic phenotype because of abnormal compo...Background In type 2 diabetes mellitus （T2DM）, high-density lipoprotein （HDL） impairs its anti-atherogenic properties and even develops to a pro-inflammatory and pro-atherogenic phenotype because of abnormal compositions and modifications. In this study, we ex- amined the effects and the related mechanisms of glycation of HDL on the proliferation and migration of vascular smooth muscle cells （VSMCs）. Methods ＆ Results Glycated HDL （G-HDL） was modified with D-glucose （25 mmol/L） in vitro. Diabetic HDL （D-HDL） was isolated from T2DM patients. Rat VSMCs were isolated from the thoracic aortas. Human VSMCs were obtained from ScienCell Research Laboratories. Alpha-actin was detected through immunofiuorescence. VSMC proliferation was assayed by Cell Count. VSMC migration was determined by transwell chamber and scratch-wound assay. Intracellular reactive oxygen species （ROS） was detected based on ROS-medi- ated 2＇,7＇-dichlorofluorescein （DCFH-DA） fluorescence. Compared to native HDL （N-HDL）, G-HDL remarkably promoted VSMC prolif- eration and migration in the dose and time-dependent manners. In addition, G-HDL enhanced ROS generation in VSMCs. However, the ROS scavenger, N-acetylcysteine, efficiently decreased ROS production and subsequently inhibited the proliferation of VSMCs induced by G-HDL. Similarly, D-HDL from T2DM patients also promoted ROS release and VSMC proliferation and migration. Conclusions HDL either glycated in vitro or isolated from T2DM patients triggered VSMC proliferation, migration, and oxidative stress. These results might partly interpret the higher morbidity of cardiovascular disease in T2DM patients.展开更多
Objective To explore the influence of oxidized high-density lipoprotein （oxHDL） on the maturation and migration of bone marrow-derived dendritic cells （BMDCs） from C57BL/6J mice. Methods The C57BL/6J mice bone ma...Objective To explore the influence of oxidized high-density lipoprotein （oxHDL） on the maturation and migration of bone marrow-derived dendritic cells （BMDCs） from C57BL/6J mice. Methods The C57BL/6J mice bone marrow cell suspension was prepared and purified. Recombinant granulocyte-macrophage colony-stimulating factor （rmGM-CSF） and recombinant interleukin-4 （rmlL-4） were used to promote monocytes to differentiate and suppress lymphocytes. Then 50μg/mL oxHDL was added to stimulate BMDCs, using 50μg/mL high-density lipoprotein （HDL） as homologous protein control, PBS as negative control, and 1 μg/mL lipopolysaccharide （LPS） as positive control. The CD86 and MHCII expression rates were detected with fluorescence-activated cell sorting （FACS）. Liquid scintillation counting （LSC） was used in mixed lymphocyte reactions （MLRs） to reflect the ability of BMDCs in stimulating the proliferation of homologous T cells, Levels of cytokines IL-12 and IL-10 were detected by ELISA. The cell migration was evaluated with the transwell system. Results Compared with PBS group, the expressions of CD86 and MHCII, counts per minute of MLRs, secretion of IL-12 and IL-10, and number of migrated cells in oxHDL group and LPS group significantly increased （all P 〈 0.05）, while the increment was less in oxHDL group than LPS group. The number of migrated cells in oxHDL group was about twice of that in HDL group. Conclusion OxHDL may promote the maturation and migration of BMDCs in vitro.展开更多
AIM: To describe the way stations of high-density lipoprotein(HDL) uptake and its lipid exchange in endothelial cells in vitro and in vivo. METHODS: A combination of fluorescence microscopy using novel fluorescent cho...AIM: To describe the way stations of high-density lipoprotein(HDL) uptake and its lipid exchange in endothelial cells in vitro and in vivo. METHODS: A combination of fluorescence microscopy using novel fluorescent cholesterol surrogates and electron microscopy was used to analyze HDL endocytosis in great detail in primary human endothelial cells. Further, HDL uptake was quantified using radio-labeled HDL particles. To validate the in vitro findings mice were injected with fluorescently labeled HDL and particle uptake in the liver was analyzed using fluorescencemicroscopy. RESULTS: HDL uptake occurred via clathrin-coated pits, tubular endosomes and multivesicular bodies in human umbilical vein endothelial cells. During uptake and resecretion, HDL-derived cholesterol was exchanged at a faster rate than cholesteryl oleate, resembling the HDL particle pathway seen in hepatic cells. In addition, lysosomes were not involved in this process and thus HDL degradation was not detectable. In vivo, we found HDL mainly localized in mouse hepatic endothelial cells. HDL was not detected in parenchymal liver cells, indicating that lipid transfer from HDL to hepatocytes occurs primarily via scavenger receptor, class B, type Ⅰ mediated selective uptake without concomitant HDL endocytosis. CONCLUSION: HDL endocytosis occurs via clathrincoated pits, tubular endosomes and multivesicular bodies in human endothelial cells. Mouse endothelial cells showed a similar HDL uptake pattern in vivo indicating that the endothelium is one major site of HDL endocytosis and transcytosis.展开更多
Sickle cell disease(SCD) is the first molecular disease in the literature. Although the structural alteration and dysfunction of the sickle hemoglobin(HbS) are well understood, the many factors modifying the clinical ...Sickle cell disease(SCD) is the first molecular disease in the literature. Although the structural alteration and dysfunction of the sickle hemoglobin(HbS) are well understood, the many factors modifying the clinical signs and symptoms of the disease are under investigation. Besides having an abnormal electrophoretic mobility and solubility, HbS is unstable. The autooxidation rate of the abnormal HbS has been reported to be almost two times of the normal. There are two more components of the oxidative damage in SCD: Free radical induced oxidative damage during vaso-occlusion induced ischemia-reperfusion injury and decreased antioxidant capacity in the erythrocyte and in the circulation. We will discuss the effects of oxidative alterations in the erythrocyte and in the plasma of SCD patients in this review.展开更多
The present study found that conditioned media from Kupffer cells preincubated with acetylated LDL or acetylated LDL and zymosan increased the number of HDL receptors on hepatocytes, using the method of conditioned me...The present study found that conditioned media from Kupffer cells preincubated with acetylated LDL or acetylated LDL and zymosan increased the number of HDL receptors on hepatocytes, using the method of conditioned media transfer. This indicated that the transferable factors produced by Kupffer cells modulate HDL receptors on hepatocytes.展开更多
目的研究多廿醇对低密度脂蛋白受体(low density lipoprotein receptor,LDL-R)活性的影响,探讨多廿醇降脂作用的机制。方法体外试验采用常规细胞培养方法,观察多廿醇对人单个核细胞LDL-R的直接作用。体内试验采用自身和组间两种对照设计...目的研究多廿醇对低密度脂蛋白受体(low density lipoprotein receptor,LDL-R)活性的影响,探讨多廿醇降脂作用的机制。方法体外试验采用常规细胞培养方法,观察多廿醇对人单个核细胞LDL-R的直接作用。体内试验采用自身和组间两种对照设计,观察多廿醇对高胆固醇血症人群LDL-R活性的影响。LDL-R活性检测选用荧光标记配体法。结果体外试验表明多廿醇在5～20mg·L-1范围内可以增强人单个核细胞LDL-R的活性,并且具有明显的量-效关系。体内试验中高胆固醇患者服用多廿醇20mg·d-14wk后,外周血单个核细胞LDL-R活性升高95%。结论多廿醇能通过增加LDL-R的活性而起降血脂作用,多廿醇降血脂是多途径的。展开更多
基金Supported by the National Natural Science Foundation of China,No. 39770164
文摘AIM: To investigate the possibility of recombinant highdensity lipoprotein (rHDL) being a carrier for delivering antitumoral drug to hepatoma cells. METHODS: Recombinant complex of HDL and aclacinomycin (rHDL-ACM) was prepared by cosonication of apoproteins from HDL (Apo HDL) and ACM as well as phosphatidylcholine. Characteristics of the rHDL-ACM were elucidated by electrophoretic mobility, including the size of particles, morphology and entrapment efficiency. Binding activity of rHDL-ACM to human hepatoma cells was determined by competition assay in the presence of excess native HDL. The cytotoxicity of rHDL-ACM was assessed by MTT method. RESULTS: The density range of rHDL-ACM was 1.063-1.210 g/mL, and the same as that of native HDL. The purity of all rHDL-ACM preparations was more than 92%. Encapsulated efficiencies of rHDL-ACM were more than 90%. rHDL-ACM particles were typical sphere model of lipoproteins and heterogeneous in particle size. The average diameter was 31.26±5.62 nm by measure of 110 rHDL-ACM particles in the range of diameter of lipoproteins. rHDL-ACM could bind on SMMC-7721 cells, and such binding could be competed against in the presence of excess native HDL. rHDL-ACM had same binding capacity as native HDL. The cellular uptake of rHDL-ACM by SMMC-7721 hepatoma cells was significantly higher than that of free ACM at the concentration range of 0.5-10 μg/mL (P<0.01). Cytotoxicity of rHDL-ACM to SMMC-7721 cells was significantly higher than that of free ACM at concentration range of less than 5 ug/mL (P<0.01) and IC50 of rHDL-ACM was lower than IC50 of free ACM (1.68 nmol/L vs3 nmol/L). Compared to L02 hepatocytes, a normal liver cell line, the cellular uptake of rHDL-ACM by SMMC-7721 cells was significantly higher (P<0.01) and in a dose-dependent manner at the concentration range of 0.5-10 μg/mL.Cytotoxicity of the rHDL-ACM to SMMC- 7721 cells was significantly higher than that to L02 cells at concentration range of 1-7.5μg/mL (P<0.01). IC50 for SMMC-7721 cells (1.68 nmol/L) was lower than that for L02 cells (5.68 nmol/L), showing a preferential cytotoxicity of rHDL-ACM for SMMC-7721 cells. CONCLUSION: rHDL-ACM complex keeps the basic physical and biological binding properties of native HDL and shows a preferential cytotoxicity for SMMC-7721 hepatoma to normal L02 hepatocytes, HDL is a potential carrier for delivering lipophilic antitumoral drug to hepatoma cells.
基金This project was supported by Grant 31200884 from the National Natural Science Foundation of China Grant 2016D016, 2016-ZQN-92, and 2016-2-75 from the Natural Science Foundation of Fujian and Grant 3502Z20154048, 3502Z20144061, and 3502Z20154047 from the Natural Scien- ce Foundation of Xiamen.
文摘Background In type 2 diabetes mellitus （T2DM）, high-density lipoprotein （HDL） impairs its anti-atherogenic properties and even develops to a pro-inflammatory and pro-atherogenic phenotype because of abnormal compositions and modifications. In this study, we ex- amined the effects and the related mechanisms of glycation of HDL on the proliferation and migration of vascular smooth muscle cells （VSMCs）. Methods ＆ Results Glycated HDL （G-HDL） was modified with D-glucose （25 mmol/L） in vitro. Diabetic HDL （D-HDL） was isolated from T2DM patients. Rat VSMCs were isolated from the thoracic aortas. Human VSMCs were obtained from ScienCell Research Laboratories. Alpha-actin was detected through immunofiuorescence. VSMC proliferation was assayed by Cell Count. VSMC migration was determined by transwell chamber and scratch-wound assay. Intracellular reactive oxygen species （ROS） was detected based on ROS-medi- ated 2＇,7＇-dichlorofluorescein （DCFH-DA） fluorescence. Compared to native HDL （N-HDL）, G-HDL remarkably promoted VSMC prolif- eration and migration in the dose and time-dependent manners. In addition, G-HDL enhanced ROS generation in VSMCs. However, the ROS scavenger, N-acetylcysteine, efficiently decreased ROS production and subsequently inhibited the proliferation of VSMCs induced by G-HDL. Similarly, D-HDL from T2DM patients also promoted ROS release and VSMC proliferation and migration. Conclusions HDL either glycated in vitro or isolated from T2DM patients triggered VSMC proliferation, migration, and oxidative stress. These results might partly interpret the higher morbidity of cardiovascular disease in T2DM patients.
基金Supported by the Foundation of Hunan Educational Committee (06C692)
文摘Objective To explore the influence of oxidized high-density lipoprotein （oxHDL） on the maturation and migration of bone marrow-derived dendritic cells （BMDCs） from C57BL/6J mice. Methods The C57BL/6J mice bone marrow cell suspension was prepared and purified. Recombinant granulocyte-macrophage colony-stimulating factor （rmGM-CSF） and recombinant interleukin-4 （rmlL-4） were used to promote monocytes to differentiate and suppress lymphocytes. Then 50μg/mL oxHDL was added to stimulate BMDCs, using 50μg/mL high-density lipoprotein （HDL） as homologous protein control, PBS as negative control, and 1 μg/mL lipopolysaccharide （LPS） as positive control. The CD86 and MHCII expression rates were detected with fluorescence-activated cell sorting （FACS）. Liquid scintillation counting （LSC） was used in mixed lymphocyte reactions （MLRs） to reflect the ability of BMDCs in stimulating the proliferation of homologous T cells, Levels of cytokines IL-12 and IL-10 were detected by ELISA. The cell migration was evaluated with the transwell system. Results Compared with PBS group, the expressions of CD86 and MHCII, counts per minute of MLRs, secretion of IL-12 and IL-10, and number of migrated cells in oxHDL group and LPS group significantly increased （all P 〈 0.05）, while the increment was less in oxHDL group than LPS group. The number of migrated cells in oxHDL group was about twice of that in HDL group. Conclusion OxHDL may promote the maturation and migration of BMDCs in vitro.
基金Supported by the Austrian Science Fund,No.P20116-B13 and No.P22838-B13
文摘AIM: To describe the way stations of high-density lipoprotein(HDL) uptake and its lipid exchange in endothelial cells in vitro and in vivo. METHODS: A combination of fluorescence microscopy using novel fluorescent cholesterol surrogates and electron microscopy was used to analyze HDL endocytosis in great detail in primary human endothelial cells. Further, HDL uptake was quantified using radio-labeled HDL particles. To validate the in vitro findings mice were injected with fluorescently labeled HDL and particle uptake in the liver was analyzed using fluorescencemicroscopy. RESULTS: HDL uptake occurred via clathrin-coated pits, tubular endosomes and multivesicular bodies in human umbilical vein endothelial cells. During uptake and resecretion, HDL-derived cholesterol was exchanged at a faster rate than cholesteryl oleate, resembling the HDL particle pathway seen in hepatic cells. In addition, lysosomes were not involved in this process and thus HDL degradation was not detectable. In vivo, we found HDL mainly localized in mouse hepatic endothelial cells. HDL was not detected in parenchymal liver cells, indicating that lipid transfer from HDL to hepatocytes occurs primarily via scavenger receptor, class B, type Ⅰ mediated selective uptake without concomitant HDL endocytosis. CONCLUSION: HDL endocytosis occurs via clathrincoated pits, tubular endosomes and multivesicular bodies in human endothelial cells. Mouse endothelial cells showed a similar HDL uptake pattern in vivo indicating that the endothelium is one major site of HDL endocytosis and transcytosis.
文摘Sickle cell disease(SCD) is the first molecular disease in the literature. Although the structural alteration and dysfunction of the sickle hemoglobin(HbS) are well understood, the many factors modifying the clinical signs and symptoms of the disease are under investigation. Besides having an abnormal electrophoretic mobility and solubility, HbS is unstable. The autooxidation rate of the abnormal HbS has been reported to be almost two times of the normal. There are two more components of the oxidative damage in SCD: Free radical induced oxidative damage during vaso-occlusion induced ischemia-reperfusion injury and decreased antioxidant capacity in the erythrocyte and in the circulation. We will discuss the effects of oxidative alterations in the erythrocyte and in the plasma of SCD patients in this review.
文摘The present study found that conditioned media from Kupffer cells preincubated with acetylated LDL or acetylated LDL and zymosan increased the number of HDL receptors on hepatocytes, using the method of conditioned media transfer. This indicated that the transferable factors produced by Kupffer cells modulate HDL receptors on hepatocytes.
文摘目的研究多廿醇对低密度脂蛋白受体(low density lipoprotein receptor,LDL-R)活性的影响,探讨多廿醇降脂作用的机制。方法体外试验采用常规细胞培养方法,观察多廿醇对人单个核细胞LDL-R的直接作用。体内试验采用自身和组间两种对照设计,观察多廿醇对高胆固醇血症人群LDL-R活性的影响。LDL-R活性检测选用荧光标记配体法。结果体外试验表明多廿醇在5～20mg·L-1范围内可以增强人单个核细胞LDL-R的活性,并且具有明显的量-效关系。体内试验中高胆固醇患者服用多廿醇20mg·d-14wk后,外周血单个核细胞LDL-R活性升高95%。结论多廿醇能通过增加LDL-R的活性而起降血脂作用,多廿醇降血脂是多途径的。
