| Chapter.l The change of plasma ANGPTL2in patients with type2diabetes mellitus and its relationship with CA-IMTObjectives:To observe the change of plasma Angptl2in patients with type2diabetes mellitus and its relationship with CA-IMT,lipids and blood glucose.Methods:99patients with type2diabetes mellitus were enrolled in this study, and another50healthy persons served as control group. Their Weight, blood pressure, fasting plasma glucose, fasting serum insulin, triglyceride, total cholesterol, high density lipoprotein, low density lipoprotein were measured, At the same time, their CA-IMT were examined by high-resolution color doppler ultrasound, Angptl2was analyzed by double antibody sandwich enzyme-linked immunos orbent assay. At last, the date was arranged for statistical analysisResults:1. Compared with normal persons, the level of Angptl2in type2diabetes was elevated,and showed a significant relation with FPG.2. Compared with control group, the level of CA-IMT in type2diabetes mellitus was higher.3. CA-IMT significantly related to Angptl2in two groups.4. In multiple stepwise regression analysislower HDL、Angptl2exihibited contributions to CA-IMT.Conclusion1. Compared with normal persons, the level of Angptl2in type2diabetes was elevated, and showed a significant relation with FPG, maybe glycimia could influence the level of Angptl22. CA-IMT was thicker in type2diabetes than that in control group, suggesting diabetes could accelerate the progress of atllerosclerosis3. Angptl2showed a positive correlation with CA-IMT,and it is CA-IMT’s mayor risk factor, suggesting that Angptl2may could lead to atherosclerosis. Chapter.2The effect of Angptl2was concerned with the apoptosis of human umbilical vein endothelial cells induced by high glucoseObjective:To investigate the effect of high glucose on the apoptosis of human umbilical vein endothelial cell and angiopoietin-like protein2expression, thus to evaluate the role of Angptl2in the high glucose-induced apoptosis in human umbilical vein endothelial cells.Methods:1. High glucose-induced endothelial cell apoptosis: HUVECs were incubated with different concentrations of glucose in (5,10,20,30Mm),5mM and30mM glucose in cultured under conditions different times (4,8,12,24,48h), in order to tetrazolium colorimetric enzyme reaction trace (MTT) cell proliferation;2. Angiopoietin-like protein2expression levels:HUVECs were incubated with different concentrations of glucose (5,10,20,30mM)and25mM mannitol, and5mM,30mM glucose and25mM mannitol in cultured under conditions different time (4,8,12,24,48h), western blot detection of Angptl2changes;3. Construction of angiopoietin-like protein2carrier.4. HUVEC cells were set in empty vector,30mM glucose group,30mM glucose+Ad-Angptl2group, Ad-Angptl2group,30mM glucose+LY294002group, Ad-Angptl2+LY294002(PI3K inhibitor) group were cultured for48hours in order to assay endothelial apoptosis through the way of TUNEL and Annexin-FITC/PI.Results:1. MTT assay showed that cell proliferation and glucose dose-effect relationship, HUVEC cells in5mM glucose under cell proliferation and30mM glucose under cell proliferation were significantly different (P<0.05); and in30mM glucose concentration under the effect of using gradient time intervention HUVEC cells found HUVEC cell proliferation and glucose effect was time-dependent,30mM glucose intervention48h group and intervention4h group compared3, proliferation significantly different (P<0.05);2. Western blot results showed that Angptl2and glucose showed dose-effect relationship, under the condition of high glucose, the expression of Angptl2is higher, they were positive correlations; compared5mM glucose with30mM glucose group,the expression of Angptl2were significantly different (P <0.05); Angptl2and glucose were showed time-dependent,the expression level of Angptl2was connect with the time of30mM glucose effect HUVEC, they were positive correlations; expression levels of Angptl2were significantly different between the time4hours and48hours under the condition of30mM glucose (P<0.05);3. By PCR and ligated product obtained after the experiment, molecular weight and sequence design are in line with expectations;4. TUNEL assay showed that, overexpression of Angpt12can induce apoptosis of HUVEC cells, while inhibiting PI3K pathway aslo can induce HUVEC cells apoptosis; compared30mM glucose group and30mM glucose+Ad-Angptl2group, the apoptosis of HUVEC cells were significantly different (P<0.05); compared30mM glucose group and30mM glucose+LY294002group, the apoptosis of HUVEC cells were significantly different (P<0.05); the results were similar between TUNEL assay and Annexin-FITC/PI.Conclusion:1. High glucose can induced the apoptosis of endothelial cells and increased the expression of Angptl2.2. Increased expression of Angptl2was positively correlated with the concentration of glucose, positive correlation with the time of high glucose.3. Overexpression of Angptl2, the rate of endothelial cell apoptosis increased, and promote the apoptosis of endothelial cells in high glucose condition.4. LY294002play an enhanced role in promoting apoptosis, high glucose and overexpression of Angptl2protein also showed the same effect with LY294002. Chapter.3Angiopoietin-like protein2induced apoptosis in HUVECs through pathway of PI3K/AktObjective:to investigate the effect of high glucose ang Angptl2transfection on the PI3K, AKT, p-PI3K, p-AKT,bax,bcl-2of phosphatidylinositol3kinase pathway (P13K/Akt).To study whether the endothelial cell apoptosis induced by high glucose was coursed by Angptl2through PI3K/Akt pathway.Methods:1.HUVEC cells were transfected six roles, such as, the empty vector,30mM glucose, transfected Ad-Angptl2, transfected with Ad-Angptl2+30mM glucose,30mM glucose+LY294002, transfected Ad-Angptl2+LY294002, and cultured for48hours in order to analysis PI3K, AKT, p-PI3K, p-AKT expression through the way of Western blot;2. HUVEC cells were transfected six roles, such as, the empty vector,30mM glucose, transfected Ad-Angptl2, transfected with Ad-Angptl2+30mM glucose,30mM glucose+LY294002transfected Ad-Angptl2+LY294002, and cultured for48hours in order to analysis bax, bcl-2protein levels using the Western blot. Results:1.Western blot results showed that:high glucose-induced HUVEC cells PI3K/AKT expression levels can be changed, while the over-expression of genes can make PI3K/AKT Angptl2change; transfected Ad-Angptl2group compared with transfected Ad-Angptl2+LY294002group and transfected Ad-Angptl2+30mM glucose group, the PI3K/AKT activation levels were significantly different (P<0.05);30mM glucose+LY294002group compared with transfected Ad-Angptl2+30mM glucose group, the PI3K/AKT activation levels were not different (P>0.05);2. Western blot assay bax and bcl-2expression showed that high glucose-induced can promote bax and bcl-2protein levels change, and overexpression of genes can make Angpt12bcl-2and bax protein expression changes; transfected Ad-Angptl2group compared with Ad-Angptl2+LY294002and Ad-Angptl2+30mM glucose group, the protein levels of bax and bcl-2were significantly different (P <0.05);Conclusion:1. The level of p-PI3k, p-Akt can be decreased under the condition of high glucose and over expression of the Angptl2, high glucose and over expression of Angptl2may prevent Akt phosphorylation, leading to increased endothelial cell apoptosis.2. LY294002is the PI3k-Akt pathway specific blocker, it can enhanced apoptosis of endothelial cells by over expression of Angptl2induced. The hindering effect was equaled between overexpression of Angptl2with the addition of LY294002.3. The expression of bax protein and the ratio of bax/bcl-2were increased under the condition of overexpression Angptl2, lead to HUVEC cells apoptosis. |
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