| ObjectiveThis study was to observe the expression of vitronectin, the situation of cytoskeleton remodeling, cell migration and blood vessels forming in HUVEC trained by high glucose. And to survey the influence of vitronectin on cytoskeleton remodeling, cell migration and blood vessels forming in HUVEC.MethodsThe first part:HUVEC were cultured respectively by normal and high glucose. The experiments were divided into group1(normal glucose group:HUVEC were trained by DMEM medium which contained25mmol/L glucose) and group2(high glucose group:HUVEC were trained by DMEM medium which contained50mmol/L glucose). Vitronectin protein expression were measured by western blot, and the micro filament cytoskeleton of HUVEC were observed by immunofluorescence cytochemical staining combined with fluorescence microscope. At last, evaluated cell migration ability with scratch wound assay and observed blood vessels forming ability with matrigel assay. These data were analysed by Independent T test sample.The second part:Disturbed the expression of vitronectin in high glucose case make use of RNA interference technology. The experiments were divided into group2(high glucose group:HUVEC were trained by DMEM medium which contained50mmol/L glucose), group3(negative interference group:HUVEC were interferenced by control SiRNA in advance, and then were trained by DMEM medium which contained50mmol/L glucose) and group4(positive interference group:HUVEC were interferenced by VN SiRNA in advance, and then were trained by DMEM medium which contains50mmol/L glucose). Repeat the experiments, incluing western bltot, immunofluorescence cytochemical staining, scratch wound assay and matrigel assay. All datas were analysed by test of homogeneity of variances first, then were conducted F test. If the differences were obvious in F test, these data were analysed by LSD of Oneway ANOVA further.ResultsThe first part:Vitronectin protein expression in group1and group2were respectively (1.098±0.085)(1.091±0.068) at0hours, and the differences were not obvious (t1,2=0.111, P1,2>0.05);After24hours, vitronectin protein expression in group1and group2were respectively (0.893±0.119)(1.295±0.080); And after48hours were respectively (0.874±0.115)(1.724±0.098), the differences between were both significant (t1,2=-4.857,-9.710, both P1,2<0.05); Immunofluorescence showed that cytoskeleton remodeling, by contrast, were even more obvious in group2after both24hours and48hours. In scratch wound assay, the cell migration ability of group2(242.000±11.130) obciously exceeded than that of group1(198.000±15.880) after24hours; And after48hours the cell migration ability of group2(334.000±10.863) was also higher than that of group1(293.000±8.681), the differences were both significant (t1,2=-5.495,-7.193, both P1,2<0.05). In matrigel assay, the number of blood vessels in group2(120.000±9.813) was much more than that of in group1(91.000±9.867) after6hours, the differences were very obvious (t1,2=-5.163, P1,2<0.05);And after12hours, the number of blood vessels in group2was (19.000±3.209), while the number of blood vessels in group1was (8.000±3.011). The differences was also obvious (t1,2=-5.659, P1,2<0.05).The second part:Vitronectin protein expression in group2,3,4were respectively (1.091±0.068)(1.002±0.029)(1.104±0.144) at0hour, the differences were not obvious (F=1.064, P>0.05);After24hours, vitronectin protein expression in group2,3,4were respectively(1.295±0.080)(1.078±0.090)(0.859±0.115), and the differences were all significant (LSD2,3=0.217, P2,3<0.05; LSD2,4=0.435, P2,4<0.05;LSD3,4=0.219, P3,4<0.05); After48hours, vitronectin protein expression in group2,3,4were respectively (1.724±0.098)(1.148±0.163)(0.693±0.167), and the differences were also significant (LSD2,3=0.576, P2,3<0.05; LSD2,4=1.031, P2,4<0.05; LSD3;4=0.455, P3,4<0.05). Immunofluorescence showed that cytoskeleton structure was most obvious in group2, was moderate in group3, and was lest obvious in group4after both24hours and48hours. In scratch wound assay, after24hours, the cell migration ability of group2,3,4were respectively (242.000±11.130)(199.000±7.055)(170.000±9.070), and the differences were significant (LSD2,3=42.833, P2,3<0.05; LSD2,4=71.333, P2,4<0.05; LSD3,4=28.500, P3,4<0.05), while after48hours the cell migration ability of group2,3,4were respectively (334.000±10.863)(265.000±22.554)(227.000±12.613), and the differences were also obvious (LSD2,3=68.667, P2,3<0.05; LSD2,4=107.500, P2,4<0.05; LSD3,4=38.833, P3,4<0.05). In matrigel assay, after6hours the number of blood vessels in group2was (120.000±9.813), in group3was (88.000±6.976), and in group4was (55.000±8.886). The differences among of them were meaningful (LSD2,3=3.283, P2,3<0.05; LSD2,4=6.567, P2,4<0.05; LSD3,4=3.283, P3,4<0.05), while the number of blood vessels in group2,3,4were respectively (19.000±3.209)(14.000±1.789)(8±3.578) after12hours, and the differences were also meaningful (LSD2,3=4.500, P2,3<0.05; LSD2,4=1.050, P2,4<0.05; LSD3,4=6.000, P3,4<0.05).Conclusions1. High glucose can bring about vitronectin expression adding, cytoskeleton remodeling, cell migration increasing and blood vessels forming enhancing.2.Disturb the expression of vitronectin can result in cytoskeleton changing, cell migration reducing and blood vessels forming lower.3. Vitronectin maybe is one of the important influence factors of diabetic retinopathy. |
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