| Objective:Atherosclerosis is the pathological basis of macroangiopathy in type 2 diabetes mellitus.Early activation of atherosclerosis is an abnormal endothelial function.One of the manifestations of endothelial dysfunction is the change in migratory function.T cell immunoglobulin and mucin-domain containing molecule(Tim3)is a glycoprotein expressed in endothelial cells.The correlation between Tim3 protein and endothelial cell migration has not been reported.The purpose of this study was to determine whether Tim3protein affects endothelial cell migration at different glucose concentrations.Methods:Human umbilical vein endothelial cells(HUVEC)were used as the research object,RNA interference(RNAi)technology was used to block the expression of Tim3 protein in endothelial cells,and cells were cultured at different glucose concentrations.In this study,experiments consisted of three groups:normal control group,si-tim3 interference group,and si-tim3 null interference group.The three groups of cells were cultured in normal medium and glucose culture medium(5.5 mmol/L glucose or 60 mmol/L glucose)for 24 h,48 h,and 72 h.Under microscope,cell morphology and cell migration were observed at different time points in each group of cells.Scratches and Transwell assays were used to assess the changes in cell migration function,and the cell mobility and migration numbers of cells cultured for 72 h were calculated.72 h cells were collected and cultured.Real-time PCR and Western blot were used to detect mRNA and protein expression levels of Tim3and marker proteins(E-cadherin,?-catenin,Vimentin,and ZEB1)related to cell migration,respectively.The 2~-??CT??CT method and the gel image processing system(Gel-Pro-Analyzer software)were performed as semi-quantitative analysis.Results:1.Cell morphology:under normal culture conditions,the cells in normal control group and Tim3 null interference group were arranged neatly and in normal morphology.However,the cells in Tim3 interference group were disordered and the proportion of irregular cells increased.Under the conditions of sugar-added medium,the cell morphology of each group was similar to that of the corresponding groups cultured in normal medium at the sugar concentration of 5.5mmol/L.When the sugar concentration was 60 mmol/L,the cell morphology of the normal cell group was also abnormal,and the disorder of the cells in the Tim3 interference group was further aggravated.2.Changes in cell migration:2.1 Scratch test:Under normal culture,the migration rate was 82.85±3.75%in the normal control group,82.57±2.15%in the ineffective interference group,and 60.37±7.86%in the Tim3 interference group,which was significantly lower than in the normal control group(P<0.05).Under the condition of sugar-added medium,the cell migration rates of normal cells group,Tim3 null interference group and Tim3 interference group were 82.34±3.52%?79.13±4.91%?65.13±4.08%respectively at the sugar concentration of 5.5mmol/L,which were consistent with those of the corresponding cells cultured in normal culture medium.At the glucose concentration of 60 mmol/L,the three groups of cell migration rates were61.36±7.42%,61.24±7.17%,and 35.58±5.25%,respectively,and the cell migration rate in the Tim3 interference group was significantly lower than that in the normal cell group(P<0.05).The mobility of the three groups of cells cultured under this condition was significantly lower than that of the corresponding groups of cells under normal culture.2.2 Transwell experiments:respond to cell migration by migrating cell numbers.Under normal culture,the number of migrating cells in the normal control group was 144±16,and the number of migrating cells in the Tim3 interference group was 53±7.There was a significant difference between the two groups(P<0.05).The number of migrating cells in the Tim3 null interference group was similar to that of the normal control group,which was148±16.Under glucose culture,when the sugar concentration was 5.5 mmol/L,the number of migrating cells in the normal cell group,the Tim3 null interference group,and the Tim3interference group was 127±14,134±20 and 52±8,respectively,which were similar to each group under normal cultivation.When the sugar concentration was 60 mmol/L,the number of migrating cells in the normal cell group was 56±8;the number of migrating cells in the Tim3 null interference group was 52±5,and the number of migrating cells in the Tim3interference group was 19±4,which was significantly less than that in the normal cell group(P<0.05).Under these conditions,the number of cell migration in the three groups was significantly lower than that in the corresponding cell group under normal culture.3.Changes of mRNA expression level of mobile marker related proteinsThe present study examined the following four proteins:E-cadherin,?-catenin,Vimentin and ZEB1.Under normal culture conditions,the expression levels of E-cadherin and?-catenin mRNA in the normal control group were 1.00±0.06 and 1.00±0.05respectively,and the expression levels of Vimentin and ZEB1 mRNA were 1.00±0.05 and1.00±0.10,respectively.Expression levels of Tim3 null interference group of the above protein mRNA were 1.13±0.07,0.93±0.06,0.98±0.05 and 0.92±0.07.The mRNA expression levels of each protein in the Tim3 interference group were 2.78±0.29,3.35±0.14,0.44±0.02,and 0.67±0.02,respectively.Compared with normal control group,the expressions of E-cadherin and?-catenin m RNA were significantly increased,while expressions of Vimentin and ZEB1 mRNA were significantly decreased(P<0.05).For glucose culture,when the glucose concentration was 5.5 mmol/L,the mRNA expression level of each protein was similar to that of the corresponding cell group under normal culture.When the glucose concentration was 60mmol/L,the mRNA expression of E-cadherin and?-catenin in normal cells were 3.25±0.11 and 4.23±0.33,respectively.The expression levels of Vimentin and ZEB1 mRNA in normal cells were 0.37±0.03 and0.49±0.03,respectively.The expression levels of E-cadherin and?-catenin mRNA in the Tim3 null interference group were 3.12±0.24 and 3.96±0.13,respectively.The expression levels of Vimentin and ZEB1 m RNA were 0.36±0.02 and 0.49±0.04,respectively,and there was no significant difference compared with the normal cell group.The expression levels of E-cadherin and?-catenin mRNA in Tim3 interference group were4.25±0.29 and 5.34±0.12,the expression levels of Vimentin and ZEB1 mRNA were 0.22±0.01 and 0.18±0.01,respectively,which were significantly different from the normal cell group(P<0.05).Compared with the corresponding cell groups under normal culture,the expression level of E-cadherin and?-catenin mRNA were significantly increased,and the expression level of Vimentin and ZEB1 mRNA were significantly decreased,and the performance of Tim3 interference group was more obvious.The difference between groups was statistically significant(P<0.05).4.Mobile markers related protein levels changeThe present study examined the following four proteins:E-cadherin,?-catenin,Vimentin and ZEB1.Under normal culture,the protein expression levels of E-cadherin and?-catenin in normal control group were 1.00±0.07 and 1.00±0.13,respectively.The expression levels of Vimentin and ZEB1 protein were 1.00±0.07 and 1.00±0.05,respectively.The expression levels of those proteins in Tim3 null interference group were0.97±0.11,1.13±0.13,1.06±0.09 and 0.93±0.09.The expression levels of those proteins in Tim3 knockdown group were 2.43±0.24,3.72±0.33,0.35±0.05,0.41±0.03.The expression levels of E-cadherin and?-catenin in Tim3 knockdown group were significantly higher than those in normal control group,while Vimentin and ZEB1 expression were decreased significantly(P<0.05).For glucose culture,the expression level of each protein was similar to that of the corresponding cell group under normal culture at a glucose concentration of5.5 mmol/L.When the glucose concentration was 60mmol/L,the expression of E-cadherin and?-catenin in normal cells were 2.34±0.11 and 3.65±0.23,respectively.The expression levels of Vimentin and ZEB1 were 0.37±0.04 and 0.40±0.04.The expression levels of E-cadherin and?-catenin in inactive interference group were 2.38±0.14 and 3.68±0.24,respectively.The expression levels of Vimentin and ZEB1 were 0.36±0.07 and 0.36±0.04,respectively,and there was no significant difference compared with the normal cell group.The expression of E-cadherin and?-catenin in Tim3 interference group were4.67±0.36 and 6.20±0.42 respectively,while the expression levels of Vimentin and ZEB1were 0.24±0.03 and 0.12±0.02,respectively,which were significantly different from those of normal cells(P<0.05).Compared with the corresponding cell groups under normal culture,the expression level of E-cadherin and?-catenin protein were significantly increased,and the expression level of Vimentin and ZEB1 protein were significantly decreased,and the performance of Tim3 interference group was more obvious.The difference between groups was statistically significant(P<0.05).Conclusion:1.Both Tim3 protein and high glucose are related to the migration function of vascular endothelial cells.In the high glucose environment or with the decrease of Tim3 protein expression,the migration capacity of endothelial cells is reduced.2.The effect of Tim3 on endothelial cell migration is independent of glucose,but there is a superposition effect between them.Blocking Tim3 protein in a high glucose environment will aggravate the damage of vascular endothelial function. |
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