【摘要】 为研究旋毛虫p53蛋白对血管内皮细胞通透性影响的相关机制，本研究以人脐静脉内皮细胞（HUVES）为细胞模型，通过体外实验利用不同浓度的旋毛虫p53重组蛋白（Ts-p53）刺激HUVES，采用CCK-8试剂盒筛选Ts-p53刺激HUVES细胞的最适浓度，通过Transwell小室方法检测内皮细胞通透性，利用荧光定量PCR(qPCR)检测细胞凋亡关键因子Bax、Bcl-2以及内皮细胞间连接蛋白Zo-1及VE-cadherin转录水平的变化，利用western blot方法检测细胞凋亡关键因子Bax、Bcl-2、Cytochrome C、Pro-Caspase-9、cl-Caspase-9、Pro-Caspase-3、cl-Caspase-3以及内皮细胞间连接蛋白Zo-1及VE-cadherin蛋白表达量的变化。结果显示，Ts-p53刺激HUVES细胞的最适浓度为60μg/mL。随着Ts-p53对HUVES细胞作用时间的增加，FITC标记的白蛋白在感染3 h、6 h时内皮通透性Pa值逐渐上升，在12 h时尤为明显，并与空白对照组相比差异极显著（P<0.01），在刺激18 h时渗出指数下降。阳性对照组为LPS刺激HUVES，该组HUVES内皮通透性Pa值约为空白对照组HUVES的1.9倍（P<0.01）。表明Tsp53可以诱导HUVES通透性的增加。qPCR和western blot结果显示，与空白对照组相比，Ts-p53刺激3 h、6 h、12 h,Bax的转录水平和表达水平呈时间依赖性增加，在刺激12 h时其转录水平和表达水平最高（P<0.01）,18 h时其转录水平和表达水平降低，阳性对照组Bax转录水平和表达水平极显著高于其他组（P<0.01）。与空白对照组比，Bcl-2的转录水平和表达水平呈时间依赖性减少，在刺激12 h时其转录水平和表达水平最低（P<0.01）,18 h时其转录水平和表达水平均增加，阳性对照组Bcl-2转录水平和表达水平均低于其他组。在刺激12 h时，Bax/Bcl-2值最低。Western blot结果显示，与空白对照组相比，Cytochrome C表达水平也随着时间增加而增高，在12 h表达量最大，18 h表达量减少，阳性对照组该蛋白表达水平高于其他组，差异极显著（P<0.01）。随着刺激时间的增加，Caspase-3和Caspase-9蛋白被活化成cl-Caspase-3和cl-Caspase-9，均在12 h时变化最为显著（P<0.01），进一步验证了细胞凋亡的发生。与空白对照组相比细胞间连接蛋白ZO-1及VE-cadherin的转录水平和表达水平均呈时间依赖性减少，在12 h表达量最少（P<0.01）。综上所述，本研究首次证实Ts-p53可以通过引发细胞凋亡、破坏内皮细胞间连接蛋白两种方式破坏内皮细胞屏障，使血管内皮细胞通透性增加，该结果为旋毛虫感染发病机制的研究奠定了基础。更多还原

【Abstract】 To study the mechanism of the effect of Trichinella spiralis p53 protein on vascular endothelial cells, human umbilical vein endothelial cells(HUVES) were used as the cell model. HUVES were stimulated with different concentrations of T. spiralis p53 recombinant protein(Ts-p53) in vitro, and the optimal concentration of Ts-p53 was screened by CCK-8 kit. The permeability of endothelial cells was detected by Transwell chamber method, while the transcriptional changes of key factors of apoptosis Bax, Bcl-2, endothelial intercellular connexin ZO-1 and VE-cadherin were detected by qPCR. The key factors of apoptosis Bax, Bcl-2, Cytochrome C, Pro-Caspase-9, cl-Caspase-9, pro-Caspase-3, cl-caspase-3, endothelial connexin ZO-1 and VE-cadherin were detected by western blot. The experimental results showed that with the increase of the incubation time of T. spiralis p53protein on HUVES, the exudation index of FITC labeled albumin gradually increased at 3 hours and 6 hours, especially high at 12hours, and there was a significant difference compared with the blank control group(P<0.01). The exudation index decreased at 18hours. When LPS stimulated HUVES in the positive control group. The positive control group was LPS stimulated HUVES, and the endothelial permeability Pa value of HUVES in this group was about 1.9 times that of blank control HUVES(P<0.01). These results showed that the p53 protein of T. spiralis could induce the increase of HUVES permeability. The results of q PCR and western blot showed that compared with the blank control group, the transcription level and expression level of Bax increased in a time-dependent manner at 3 hours, 6 hours and 12 hours(P<0.01). The transcription level and expression level were the highest at12 hours and decreased at 18 hours(P<0.01). The transcription level and expression level of positive control group were significantly higher than those of other groups(P<0.01). Compared with the blank control group, the transcription level and expression level of Bcl-2 decreased in a time-dependent manner. The transcription level and expression level of Bcl-2 were the lowest at 12 hours of stimulation and increased at 18 hours(P<0.01). The transcription level and expression level of positive control group were lower than those of other groups. The change of Bax/Bcl-2 was the most significant at 12 hours of stimulation.The results of western blot showed that compared with the control group, the expression level of Cytochrome C increased with time(P<0.01). The expression level was the highest at 12 hours and decreased at 18 hours(P<0.01). The expression level of the positive control group was significantly higher than that of other groups(P<0.01). With the increase of stimulation time, Caspase-3 and Caspase-9 proteins were activated and turned to cl-Caspase-3 and cl-Caspase-9. This change was the most significant at 12 hours(P<0.01). Intercellular connexin ZO-1 and VE-cadherin decreased in a time-dependent manner at both transcription and expression levels, with the least expression at 12 hours. In conclusion, Ts-p53 can destroy the endothelial cell barrier and increase the permeability of vascular endothelial cells by inducing apoptosis and destroying the connexin between endothelial cells.更多还原