Study About The Glucose Density Intermittence Caused Vascular Endothelial Cell Damage Degree And Its Pathogenesis

Diabetes mellitus(DM)is a kind of metabolic dysfunction characterized by hyperglycemia which is caused by various pathogenesis.It-is always associated with macrovascular complications such as atherosclerosis(AS).Hyperglycemia is the symbol of DM,and also the major factor that results in chronic vascular complications in DM.The pathological effects exist in two ways;chronic intermittent hyperglycemia and chronic persistent hyperglycemia.Chronic intermittent hyperglycemia means a status of chronic intermittent or paroxysmal high level of blood glucose,which is also named as drift of glucose.Recent studies have found that the former one has the priority to accelerate the happening and development of macrovascular complications in patients with DM,which has captured the attention of experts in the field of the prevention and treatment of mac.rovasculopathy.Until now the research results in this field have been reported in foreign countries.However,it is relatively fewer in our country and most are clinical observational researches.In this research we are to explore the injuries of glucose drift on vascular endothelial cells and the pathogenesis of drift of glucose to cause atherosclerosis from the basic field.It will be realized through the observation of the apoptosis,inhibitory rate of cell survival and morphology changes of the cultivated endothelial cells in human umbilical vein that is in different concentration of glucose.It has been certified that hyperglycemia of type 2 DM,especially glucose fluctuation could damage the cardiovascular endothelial cells.Vascular endothelial cells are not only the barrier of endothelium but also regarded as the biggest endocrine organ for its function of secretion.It is one of the pathogenesis of atherosclerosis that under the situation of glucose fluctuation,the functions of endothelial cells are changed,which leads to the changes of synthesis and nature of some cytokines. Nowadays it is thought that AS is a disease based on inflammation.Numerous researches home and abroad have found that transforming growth factorβ1(TGF-β1) has a crucial impact on the "happening of atherosclerosis,but the changes of its expression and secretion in the condition of fluctuating glucose have not yet been reported.And our research will reveal it to explore the significance of fluctuating glucose in the happening and development of atherosclerosis.Atherosclerosis(AS)is the macrovasculopathy of type 2 DM,which is also the most common complication of type 2 DM.Recent studies have found that MMP-9,as a kind of proteinase that can degrade extracellular matrix,can accelerate the forming,development and rupture of atheromatous plaque.Increased glucose can induce its expression which would increase in disease of AS.But the reports about the expression of MMP-9 in the condition of intermittent hyperglycemia are few.We test the changes of MMP-9 mRNA expression and its protein level by applying the technology of molecular biology,to explore the changes of MMP-9 expression affected by the drift of glucose,and our researches are aimed to explore how drift of glucose works in the progress of this macrovasculopathy in the disease of type 2 DM.Methods1,.culture and counting of cellsVascular endothelial cell lines 304(VEC304)and THP-1 were purchased from CCTCC(China Center for Type Culture Collection).Cultures were maintained at 37℃,and we would shake the frozen VEC304 to thaw it out as soon as posssilble.Then we incubated it with DMEM culture media at 37℃in air.containing 5%CO₂.The culture media was changed every two days and the mixture of 0.125%trypsin and 0.02%E D T A was used to digest and transfer of culture.Cells were incubated in 50ml culture bottles and on 6 pore and 96 pore culture boards until the cells covered the whole container.2,Experimental groupsWe incubated the above cells in another media with 1%FBS(fetal bovine serum) for 24 hours to make the cells in phase GO/G1.Then we divide them into four groups;â‘ group of 5mmol/L glucose;â‘¡group of 20mmol/Lglucose;â‘¢group of altemating5mmol/Land20mmol/L glucose every 24 hours.â‘£group of 20mmol/ L mannitol.The following cuture would be maintained at 37℃in air containing 5%CO2 and last 14days.3,MTT method to determine the cell suivival rateAfter inputing 20μl MTT into each hole of the 96 pore culture board mentioned above in the condition of 37℃and incubating for another 4 hours,we absorbed the serum,inputed 150μl DMSO and shaked it for 10rain.And then we measured the light absorption value of each hole in spectrum of 490nm with enzyme-linked immunometer. The cell survival rate= mean OD of the experimental group/mean OD of the control group×100%.4,Mensuration of cell apoptosisThe apoptosis was studied by flow cytometric Annexin V-FITC/PI dual labeling technique.After washed by PBS once,by applying 1×suspention cells of binding buffer until 1×10⁶/ml.Diluting.100μl(1×10⁵)to 5ml,then put 5μl Annexin V-FITC and 10μl PI well mixed,at room temperature keep away from light for 15min.Put in 400μl 1×binding buffer each.Then checked on machine.5,AO(Acridine orange)cell fluorescence stainGet the cultivated cells that have cultured above and washed by PBS for four times 95%ethyl alcohol,1%acetic acid works 30sen,input 0.01%AO dying for 5rain and washed by PBS for 1min,0.1mmol/L CaCl₂ for 2min to distinguish the colors,and washed by PBS for three times,Glyceral;PBS 1;1 airproofed.Put under the filter of the fluorescence microscope and check instantaneously.6,Examination of mRNA expression with RT-PCR method We distilled the RNA of cells with Trizol method to determine the value of OD₂₆₀/OD₂₈₀and the concentration of RNA.2μgRNA was reverse-trancripted to cDNA,and we would do the PCR enhancing of 2ul cDNA.7,Examination of the content of TGFβ1An ELISA kit(Boehringer Mannheim,Mannheim,Germany),which quantitatively detects cytosolic histone-associated DNA fragments,was employed to assess apoptosis in adhered HUVECs.HUVEC DNA fragments were measured according to the proceduresdescribed in the ELISA kit.Briefly,the cytosolic fraction(13000-g supematant)of HUVECs was used as antigen source in a sandwich ELISA with a primary anti-histone monoclonal antibody coated to the microtiter plate and a second anti-DNA monoclonal antibody coupled to per0xidase.The percentage of DNA fragmentation was calculated according to the following formula;%DNA framents=[(OD-stimulated cells - OD blank)/(OD control cells - OD blank)]×100, where OD is optical density.8,Immunohistochemistry examination of TGFβ130%hydrogen peroxide and pure methanol(1;50)were mixed,then sections were soaked for 30min at room temperature.Normal goat serum blocking solution was added for 20min at room temperature after washed by distilled water(3×5').20μl rabbit anti human TGFβ1 polyclonal antibody(1;50)was added to stay overnight at 4℃on every section.Biotined goat anti rabbit IgG was added for 20min at 20℃after washed by distilled water(3×5')after washed by distilled water(3×5').SABC was added for 20min at 20℃after washed by distilled water(3×5')after washed by distilled water(3×5').DAB was used after washed by distilled water(3×5'),then sections were stained by hematoxylin and covered With neutral gum. 9,Quantitive examination in Western blotEndothelial cell were cultured in 250ml culture bottles and collected at different time,then proteins were extracted'.Equal amounts of protein in every exponent were subjected to electrophoresis on a 10%SDS-PAGE and transferred onto nitrocellulose membrane.After washing membrane,blocking,hybridization,the protein were revealed by ECL chemiluminescence system.Quantative analysis of MMP-9 protein was carried out by using America Chemin Imager 5500 electrophoresis gel image analyzer.10,Statistical analysisWe use x+s to express all the data.We used t analysis to compare the significance of difference between groups,p<0.05 was considered statistically significant.The data were analyzed using the statistical software SPSS12.0.Results1,The variable inhibitory rate of cell survival effected by different concentration of glucose.After 14 days' cultivation,the cell survival rate of 4 groups vary obviously. As a comparative group,20mmol/L glucose group is 85%lower than 5mmol/L glucose group;however,the fluctuating 5/20mmol/L group is 43%lower than the 2 groups mentioned above,its survival rate declines dramatically with P＜0.05.2,The result of examination of apoptosis with flow cytometry(FCM) This kind of cell has a certain apoptosis ratio,and after managed by the different density of glucose,by applying stained which can make AnnexinV-FITC propidium iodide(PI),to detect cell apoptosis can reveal the apoptosis more accurately and objectively.We can separate the cells into the following 4 groups;AnexinV-FITC-/PI-, normal cells;AnnexinVFITSC+/PI-,early apoptosis cells;AnnexinVFITC+/PI+, apoptosis cell;AnnexinV-FITC-/PI+,harm cell.In the 2D coordinates,the x-axis shows the FITC signal intensity,and the y-axis shows the PI signal intensity.Hence the Quadrantâ…¡is harm cell,Quadrantâ…¢is normal cells,Quadrantâ… is apoptosis cell, Quadrantâ…£is early apoptosis cell.After 14 days' cultivation in the conditions of 4 different glucose density,by using staine of AnnexinV-FITC-/PI which to mark the apoptosis cells,the results,analyzed by FCM,are illustrated in Figure 2,3,4.Repeat this process for 3 times,and the statistics shows the ratio of vascular endothelial cell apoptosis,which is expressed in the way of the average number + standard subtraction SD.Contrasted with the normal-density comparative group,the apoptosisratio of 20mmol/L glucose group is 19.6;5/20mmol/L is 29.11;5/20mmol/L group is make more content apparently,P＜0.05.3,AO(Acridine orange)cell fluorescence stainAO has the appetency with DNA and RNA within a cell.But the colors emit fluorescence after compounded.DNA emits light green while RNA shows red.The number of cell in 5mmol/L group is large,stained emits light green,and the cell's configuration is normal.The configuration turns smaller in 5/20 mmol/L group, scanning darkles;number declines.4,Examination ofmRNA expression with RT-PCR methodThe TGF-β1 and Bax mRNA expressions of Group 5mmol/L and 20mmol/L increase more than Group 5/20mmol/L through RT-PCR detection.But the mRNA expressions of Bcl-2 decrease obviously,P＜0.05,and there is no evident difference between Group 5mmol/L and Group 20mmol/L,P＞0.05.5,The protein level of MMP-9 in VEC304.In this experiment the MMP-9 protein Within glucose-cultivated group -- density is 5/20mmol/L-- went up(P＜0.05).Conclusion1,Hyperglycemia can induce apoptosis of vascular endothelial cell,however, intermittent hyperglycemia can lead to higher apoptosis ratio of the cultivated human vascular endothelial cell.Therefore,intermittent hyperglycemia is more dangerous than ralative stable hyperglycemia.2,Comparing with narmal serum glucose and persistent hyperglycemia, fluctuating hyperglycemia can make more content of TGF-β1 protein secreted by human vascular endothelial cells and more mRNA expressions of TGF-β1,and as we have known,TGF-β1 has an important effect on atherosclerosis.Above all,fluctuating hyperglycemia can accelerate the advancement of atherosclerosis.3,One of the reason that intermittent hyperglycemia make more content of MMP-9 protein secreted by human vascular endothelial cell and more mRNA expression of MMP-9 maybe relate to the unstable plaque of atherosclerosis.