To quantitatively analyze apoptotic and secondary necrotic cells unde r apoptosis conditions. Methods. The cells of Burkitt lymphoma (BL) cell line Raji were incubated with 1 .0 μmol/L dexamethasone (DEX) for 2, 4 an...To quantitatively analyze apoptotic and secondary necrotic cells unde r apoptosis conditions. Methods. The cells of Burkitt lymphoma (BL) cell line Raji were incubated with 1 .0 μmol/L dexamethasone (DEX) for 2, 4 and 8 h respectively, then stained with Annexin V FITC (fluorescein isothiocyanate conjugated) which was used to detec t the exposed phosphatidylserine (PS) on the epimembrane resulting from a loss o f phospholipid asymmetry in the early stage of apoptosis, and also stained with propidium iodide (PI) which allows analysis of secondary necrotic cells related with cell membrane and DNA damage that probably represent late stage of apoptosi s, then apoptotic cells were quantified by flow cytometry (FCM). Furthermore, An nexin+/PI and Annexin+/PI+ cells were sorted by fluoresence activated cell sorter (FACS), and identified by electron microscopy (EM) and DNA gel electroph oresis. Results. The percentage of apoptotic cells was found to increase with the incuba tion time (r=0.97). This method was sensitive with low detection limit (0.02%), and was reproducible with low coefficient variance (CV)(4.2%). Meanwhile, the Annexin+/PI and Annexin+/PI+ cells were identified as apoptotic and necroti c cells under EM, and DNA extracted from the Annexin+/PI cells was characteri zed by “ladder pattern”. Conclusions. Annexin V assay is a specific, sensitive, accurate, reproductive and quantitative method for analyzing apoptotic cells.展开更多
AIM： To characterise the effect of energy restriction （ER） on liver lipid and primary metabolite profile by using metabolomic approach. We also investigated whether the effect of energy restriction can be further e...AIM： To characterise the effect of energy restriction （ER） on liver lipid and primary metabolite profile by using metabolomic approach. We also investigated whether the effect of energy restriction can be further enhanced by modification of dietary protein source and calcium. METHODS： Liver metabolomic profile of lean and obese C57BI/6J mice （n = 10/group） were compared with two groups of weight-reduced mice. ER was performed on control diet and whey protein-based high-calcium diet （whey ＋ Ca）. The metabolomic analyses were performed using the UPLC/MS based lipidomic platform and the HPLC/MS/MS based primary metabolite platform.RESULTS： ER on both diets significantly reduced hepatic lipid accumulation and lipid droplet size, while only whey ＋ Ca diet significantly decreased blood glucose （P 〈 0.001） and serum insulin （P 〈 0.01）. In hepatic lipid species the biggest reduction was in the level of triacylglycerols and cerarnides while the level of cholesterol esters was significantly increased during ER. Interestingly, diacylglycerol to phospholipid ratio, an indicator of relative amount of diabetogenic diglyceride species, was increased in the control ER group, but decreased in the whey ＋ Ca ER group （P 〈 0.001, vs obese）. ER on whey ＋ Ca diet also totally reversed the obesity induced increase in the relative level of lipotoxic cerarnides （P 〈 0.001, vs obese; P 〉 0.05, vs lean）. These changes were accompanied with up-regulated TCA cycle and pentose phosphate pathway rnetabolites. CONCLUSION： ER-induced changes on hepatic rnetabolornic profile can be significantly affected by dietary protein source. The therapeutic potential of whey protein and calcium should be further studied.展开更多
Background Circulating microparticles （MPs） have been reported to be associated with coronary artery disease （CAD）. In this study, we explored the relationship between MPs procoagulant activity and characteristics...Background Circulating microparticles （MPs） have been reported to be associated with coronary artery disease （CAD）. In this study, we explored the relationship between MPs procoagulant activity and characteristics of atherosclerotic plaque detected by 64-slice computed tomography angiography （CTA）. Methods In 127 consecutive patients with CAD but without acute coronary syndrome and who under went 64-slice CTA, MPs procoagulant activity in plasma Coy a thrombin generation test）, soluble form of lectin-like oxidized low-density lipoprotein receptor-1 （sLOX-1） and N（epsilon）-（carboxymethyl） lysine （CML） circulating levels （by ELISA） were measured. A quantitative volumetric analysis of the lumen and plaque burden of the vessel wall （soft and calcific components）, for the three major coronary vessels, was performed. The patients were classified in three groups according to the presence of calcium volume： non-calcified plaque （NCP） group （calcium volume （%） = 0）, moderate calcified plaque （MCP） group （0 〈 calcium volume （%） 〈 1）, and calcified plaque （CP） group （calcium volume （%） 〉 1）. Results MPs procoagulant activity and CML levels were higher in MCP group than in CP or NCP group （P = 0.009 and P = 0.027, respectively）. MPs procoagulant activity was positively associated with CML （r = 0.317, P 〈 0.0001） and sLOX-1 levels （r = 0.216, P = 0.0025）. Conclusions MPs procoagulant activity was higher in the MCP patient group and correlated positively with sLOX-1 and CML levels, suggesting that it may characterize a state of blood vulnerability that may locally precipitate plaque instability and increase the risk of subsequent major cardiovascular events.展开更多
文摘To quantitatively analyze apoptotic and secondary necrotic cells unde r apoptosis conditions. Methods. The cells of Burkitt lymphoma (BL) cell line Raji were incubated with 1 .0 μmol/L dexamethasone (DEX) for 2, 4 and 8 h respectively, then stained with Annexin V FITC (fluorescein isothiocyanate conjugated) which was used to detec t the exposed phosphatidylserine (PS) on the epimembrane resulting from a loss o f phospholipid asymmetry in the early stage of apoptosis, and also stained with propidium iodide (PI) which allows analysis of secondary necrotic cells related with cell membrane and DNA damage that probably represent late stage of apoptosi s, then apoptotic cells were quantified by flow cytometry (FCM). Furthermore, An nexin+/PI and Annexin+/PI+ cells were sorted by fluoresence activated cell sorter (FACS), and identified by electron microscopy (EM) and DNA gel electroph oresis. Results. The percentage of apoptotic cells was found to increase with the incuba tion time (r=0.97). This method was sensitive with low detection limit (0.02%), and was reproducible with low coefficient variance (CV)(4.2%). Meanwhile, the Annexin+/PI and Annexin+/PI+ cells were identified as apoptotic and necroti c cells under EM, and DNA extracted from the Annexin+/PI cells was characteri zed by “ladder pattern”. Conclusions. Annexin V assay is a specific, sensitive, accurate, reproductive and quantitative method for analyzing apoptotic cells.
基金Foundation for Nutrition Research, Academy of Finland, Sigrid Juselius Foundation and Valio Ltd., Helsinki, Finland
文摘AIM： To characterise the effect of energy restriction （ER） on liver lipid and primary metabolite profile by using metabolomic approach. We also investigated whether the effect of energy restriction can be further enhanced by modification of dietary protein source and calcium. METHODS： Liver metabolomic profile of lean and obese C57BI/6J mice （n = 10/group） were compared with two groups of weight-reduced mice. ER was performed on control diet and whey protein-based high-calcium diet （whey ＋ Ca）. The metabolomic analyses were performed using the UPLC/MS based lipidomic platform and the HPLC/MS/MS based primary metabolite platform.RESULTS： ER on both diets significantly reduced hepatic lipid accumulation and lipid droplet size, while only whey ＋ Ca diet significantly decreased blood glucose （P 〈 0.001） and serum insulin （P 〈 0.01）. In hepatic lipid species the biggest reduction was in the level of triacylglycerols and cerarnides while the level of cholesterol esters was significantly increased during ER. Interestingly, diacylglycerol to phospholipid ratio, an indicator of relative amount of diabetogenic diglyceride species, was increased in the control ER group, but decreased in the whey ＋ Ca ER group （P 〈 0.001, vs obese）. ER on whey ＋ Ca diet also totally reversed the obesity induced increase in the relative level of lipotoxic cerarnides （P 〈 0.001, vs obese; P 〉 0.05, vs lean）. These changes were accompanied with up-regulated TCA cycle and pentose phosphate pathway rnetabolites. CONCLUSION： ER-induced changes on hepatic rnetabolornic profile can be significantly affected by dietary protein source. The therapeutic potential of whey protein and calcium should be further studied.
文摘Background Circulating microparticles （MPs） have been reported to be associated with coronary artery disease （CAD）. In this study, we explored the relationship between MPs procoagulant activity and characteristics of atherosclerotic plaque detected by 64-slice computed tomography angiography （CTA）. Methods In 127 consecutive patients with CAD but without acute coronary syndrome and who under went 64-slice CTA, MPs procoagulant activity in plasma Coy a thrombin generation test）, soluble form of lectin-like oxidized low-density lipoprotein receptor-1 （sLOX-1） and N（epsilon）-（carboxymethyl） lysine （CML） circulating levels （by ELISA） were measured. A quantitative volumetric analysis of the lumen and plaque burden of the vessel wall （soft and calcific components）, for the three major coronary vessels, was performed. The patients were classified in three groups according to the presence of calcium volume： non-calcified plaque （NCP） group （calcium volume （%） = 0）, moderate calcified plaque （MCP） group （0 〈 calcium volume （%） 〈 1）, and calcified plaque （CP） group （calcium volume （%） 〉 1）. Results MPs procoagulant activity and CML levels were higher in MCP group than in CP or NCP group （P = 0.009 and P = 0.027, respectively）. MPs procoagulant activity was positively associated with CML （r = 0.317, P 〈 0.0001） and sLOX-1 levels （r = 0.216, P = 0.0025）. Conclusions MPs procoagulant activity was higher in the MCP patient group and correlated positively with sLOX-1 and CML levels, suggesting that it may characterize a state of blood vulnerability that may locally precipitate plaque instability and increase the risk of subsequent major cardiovascular events.
