肺炎衣原体感染通过促使VE钙粘素磷酸化促进VE钙粘素内吞而增加血管内皮细胞通透性

发布时间：2018-06-01 16:31

本文选题：肺炎衣原体 + 血管内皮细胞　；参考：《天津医科大学》2017年硕士论文

【摘要】：利用肺炎衣原体(Chlamydia pneumoniae,C.pn)体外感染人脐静脉内皮细胞系EA.hy926细胞模型,观察C.pn感染对血管内皮细胞(Vascular endothelial cell,VEC)通透性的影响,并从血管内皮细胞钙粘素(Vascular endothelial cadherin,VE cadherin)内吞的角度探讨C.pn感染增加VEC通透性的机制,证实C.pn感染可通过促使VE钙粘素磷酸化促进VE钙粘素内吞而增加VEC通透性。方法:1.免疫荧光法检测VEC生长致密时的细胞间连接;2.测量累计荧光透过率检测C.pn感染对VEC通透性的影响;3.CCK-8实验检测氯喹在作用时间和工作浓度内对VEC的毒性;4.用氯喹处理VEC后,采用免疫荧光法观察C.pn感染后对VE钙粘素内吞的影响;5.CCK-8实验检测血管内皮生长因子(Vascular endothelial growth factor,VEGF)在作用时间和工作浓度内对VEC的毒性;6.应用Western blot实验检测C.pn感染VEC后VE钙粘素总蛋白表达水平的变化;7.利用质膜提取试剂盒提取VEC细胞膜蛋白或以胰酶去除细胞膜后提取VEC胞浆蛋白,应用Western blot实验观察C.pn感染后VE钙粘素的内吞情况;8.CCK-8实验检测内吞抑制剂氯丙嗪在作用时间和工作浓度内对VEC的毒性;9.测量累计荧光透过率检测加入氯丙嗪后,C.pn感染对VEC通透性的影响;10.CCK-8实验检测缓激肽在作用时间和浓度内对VEC的毒性;11.应用Western blot实验检测Src激酶抑制剂PP2对C.pn感染促进VE钙粘素Y658位点磷酸化的影响;12.测量累计荧光透过率观察PP2对C.pn感染增加VEC通透性的影响;13.利用质膜提取试剂盒提取VEC细胞膜蛋白或以胰酶去除细胞膜后提取VEC胞浆蛋白,应用Western blot实验检测PP2对C.pn感染促进VE钙粘素内吞的影响。结果:1.光学显微镜下可见,生长致密的VEC细胞间连接完整;2.C.pn感染VEC 0 h、18 h、24 h、36 h、48 h后,VEC通透性明显增高,其中以感染24 h后增高最为明显,差异具有统计学意义(P0.05);3.CCK-8实验结果显示,以20μmol/l、40μmol/l、60μmol/l和80μmol/l工作浓度的氯喹处理VEC 24 h对VEC活力无明显影响,差异无统计学意义,而以100μmol/l的氯喹处理VEC 24 h后,VEC活力明显下降,差异有统计学意义(P0.05);4.激光共聚焦结果显示,正常VEC内呈特征性荧光绿色的VE钙粘素均匀分布于细胞与细胞连接处。C.pn感染24 h后,VE钙粘素在VEC胞膜处表达明显减少,但在胞浆中表达增多,且呈弥散分布,细胞间连接不再清晰分明,并出现明显缝隙。而氯喹处理后,C.pn感染引起的VE钙粘素由胞膜向胞浆内分布即VE钙粘素内吞更为明显。5.CCK-8实验结果显示,在工作时间内,工作浓度的VEGF对VEC活力无明显影响,差异无统计学意义(P0.05);6.Western blot结果显示:C.pn感染组、VEGF组、C.pn感染+氯喹处理组、VEGF+氯喹处理组以及氯喹处理组与正常对照组相比,VE钙粘素总蛋白表达水平均无明显变化(P0.05);7.Western blot结果显示:C.pn感染组和VEGF处理组的细胞膜VE钙粘素表达水平与正常对照组明显减少,差异具有统计学意义(P0.05),而正常对照组细胞浆中仅能检测到少量的VE钙粘素,C.pn感染组与VEGF处理组的细胞浆中VE钙粘素较正常对照组有所增加;应用溶酶体抑制剂氯喹抑制内吞的VE钙粘素降解后,C.pn感染+氯喹处理组与C.pn感染组相比,其细胞膜上VE钙粘素表达水平无明显差异,但前者细胞浆中VE钙粘素表达水平较后者有所增加,差异具有统计学意义(P0.05);VEGF+氯喹处理组与VEGF处理组的VEC细胞膜上VE钙粘素表达水平无明显增加,但与后者相比,前者细胞浆中VE钙粘素表达水平明显增加,差异具有统计学意义(P0.05),提示C.pn感染可明显促进VE钙粘素内吞。8.CCK-8实验结果显示,在工作时间内,工作浓度的氯丙嗪对VEC活力无明显影响,差异无统计学意义(P0.05);9.通过测量累计荧光透过率发现,加入氯丙嗪抑制VE钙粘素内吞后,C.pn感染引起的VEC通透性增加被明显抑制(P0.05);10.CCK-8实验结果显示,在工作时间内,工作浓度的缓激肽对VEC活力无明显影响,差异无统计学意义(P0.05);11.Western blot结果显示:C.pn感染组和缓激肽处理组VE钙粘素磷酸化水平较正常对照组显著增加,差异有统计学意义(P0.05);PP2+C.pn感染组的VE钙粘素磷酸化水平较C.pn感染组明显下降,差异有统计学意义(P0.05);12.累计荧光透过率检测结果显示,PP2预处理可削弱缓激肽促进VEC通透性增加的作用,差异具有统计学意义(P0.05);同时,PP2预处理VEC后,C.pn感染增加VEC通透性的作用也明显减弱,差异具有统计学意义(P0.05),提示C.pn感染可通过促进VE钙粘素磷酸化而增加VEC通透性;13.Western blot结果显示,C.pn感染组和缓激肽处理组的细胞膜VE钙粘素磷酸化水平与与正常对照组相比明显增加,差异有统计学意义(P0.05);而PP2+C.pn感染组的VE钙粘素磷酸化水平较C.pn感染组明显减少,差异有统计学意义(P0.05);且在细胞浆中,C.pn感染组和缓激肽处理组的VE钙粘素磷酸化水平较正常对照组明显增加,差异有统计学意义(P0.05);C.pn感染+氯喹处理组的VE钙粘素Y658位点的酪氨酸磷酸化水平较C.pn感染组明显增加,差异有统计学意义(P0.05)。结论:C.pn感染可能通过促进VE钙粘素Y658位点的磷酸化促进VE钙粘素内吞,进而增加VEC通透性。
[Abstract]:The effect of C.pn infection on the permeability of vascular endothelial cells (Vascular endothelial cell, VEC) was observed by using Chlamydia pneumoniae (C.pn) and EA.hy926 cell model in human umbilical vein endothelial cell line in vitro. The mechanism of increasing the permeability of VEC confirmed that C.pn infection could increase VEC permeability by promoting VE calcium phosphate phosphorylation to promote VE cadherin endocytosis. Method: 1. immunofluorescence was used to detect the intercellular connection of VEC in the growth and density, and the cumulative fluorescence transmittance was used to detect the effect of C.pn infection on the permeability of VEC, and the 3.CCK-8 test was used to detect chloroquine. The toxicity of VEC in time and working concentration; 4. after VEC was treated with chloroquine, the effect of C.pn infection on the endocytosis of VE cadherin was observed by immunofluorescence; 5.CCK-8 test was used to detect the toxicity of vascular endothelial growth factor (Vascular endothelial growth factor, VEGF) to VEC in action time and working concentration; 6. The changes in the total protein expression level of VE cadherin after VEC infection after C.pn infection; 7. using the plasma membrane extraction kit to extract VEC cell membrane protein or to remove the VEC cytoplasm after removal of the cell membrane by trypsin, and to observe the endocytosis of VE cadherin after C.pn infection by Western blot test. The 8.CCK-8 test was used to detect the time and work of the endocytosis inhibitor chlorpromazine. Toxicity of VEC in concentration; 9. the cumulative fluorescence transmittance was measured to detect the effect of C.pn infection on VEC permeability after chlorpromazine was added; 10.CCK-8 test detected the toxicity of bradykinin to VEC in action time and concentration; 11. the Western blot test was used to detect the effect of Src kinase inhibitor PP2 on C.pn infection promoting the phosphorylation of VE cadherin. Ringing; 12. measured the cumulative fluorescence transmittance to observe the effect of PP2 on the increase of VEC permeability to C.pn infection; 13. using a plasma membrane extraction kit to extract VEC cell membrane protein or the removal of VEC cytoplasm with pancreatin, and the effect of PP2 on C.pn infection promoting VE calcium endocytosis by Western blot test. Results: 1. optical microscope can be used. It was found that the dense VEC cells were connected completely, and the 2.C.pn infection of VEC 0 h, 18 h, 24 h, 36 h, and 48 h increased obviously, and the increase was most obvious after infection 24 h, and the difference was statistically significant (P0.05). The 3.CCK-8 experiment results showed that chloroquine, with 20 mu, 40 mu, 60 mu and 80 mu working concentration, treated 24 There was no significant difference in vitality, but the activity of VEC decreased significantly after treating VEC 24 h with 100 mol/l chloroquine, and the difference was statistically significant (P0.05). 4. laser confocal results showed that the VE cadherin in normal VEC with characteristic fluorescent green was distributed uniformly at the cell and cell junction.C.pn infection 24 h, VE cadherin was in VEC The expression of the cell membrane was significantly reduced, but the expression in the cytoplasm was increased and distributed, and the intercellular connection was no longer clear and clear. And after chloroquine treatment, the VE cadherin caused by C.pn infection was more obvious by the intracellular distribution of the cytoplasm in the cytoplasm, that is, the VE cadherin endocytosis, and the working time, the working concentration. There was no significant effect of VEGF on VEC activity, and the difference was not statistically significant (P0.05). 6.Western blot results showed that C.pn infection group, VEGF group, C.pn infection + chloroquine treatment group, VEGF+ chloroquine treatment group and chloroquine treatment group compared with normal control group, VE cadherin total protein expression level had no significant changes (P0.05); 7.Western conclusions showed that: The expression of VE calcalin in the cell membrane of the infection group and the VEGF treatment group was significantly decreased, the difference was statistically significant (P0.05), while the normal control group was only able to detect a small amount of VE calcalin, and the VE calcium gluin in the C.pn infection group and the VEGF treatment group was increased than that in the normal control group, and the lysosome inhibition was used. After the degradation of endocytic VE cadherin by chloroquine, there was no significant difference in the expression level of VE cadherin on the cell membrane of C.pn infection + chloroquine treatment group and C.pn infection group, but the expression level of VE cadherin in the former cytoplasm was significantly higher than that in the latter, and the difference was statistically significant (P0.05); VEC cells in VEGF+ chloroquine treatment group and VEGF treatment group were in VEC cells. The expression level of VE cadherin on the membrane was not significantly increased, but compared with the latter, the expression level of VE cadherin in the former cytoplasm was significantly increased, the difference was statistically significant (P0.05), suggesting that C.pn infection could obviously promote the results of VE endocytosis.8.CCK-8 experiment, and the working concentration of chlorpromazine had no significant effect on the activity of VEC in the working time. The difference was not statistically significant (P0.05); 9. by measuring the cumulative fluorescence transmittance, the increase of VEC permeability caused by C.pn infection was significantly inhibited after the addition of chlorpromazine to the VE cadherin endocytosis (P0.05). The results of 10.CCK-8 experiment showed that the working concentration of bradykinin had no significant effect on the activity of VEC in the working time, and there was no significant difference between them. P0.05); the results of 11.Western blot showed that the phosphorylation level of VE cadherin in the C.pn infection group and the bradykinin treatment group was significantly higher than that in the normal control group, and the difference was statistically significant (P0.05); the VE cadherin phosphorylation level in PP2+C.pn infection group was significantly lower than that in the C.pn infection group, and the difference was statistically significant (P0.05); 12. cumulative fluorescence transmission rate detection junction was found. The results showed that PP2 pretreatment could weaken the effect of bradykinin on promoting the increase of VEC permeability, and the difference was statistically significant (P0.05). At the same time, after PP2 pretreatment VEC, the effect of C.pn infection on VEC permeability increased obviously, the difference was statistically significant (P0.05), suggesting that C.pn infection could increase VEC permeability by promoting the phosphorylation of VE cadherin; 13. Western blot results showed that the level of VE cadherin phosphorylation in the cell membrane of the C.pn infection group and the bradykinin treatment group increased significantly compared with the normal control group, and the difference was statistically significant (P0.05), while the level of the phosphorylation of VE cadherin in the PP2+C.pn infected group was significantly less than that in the C.pn infection group, and the difference was statistically significant (P0.05); and in the cytoplasm, C, C, C, and C. The phosphorylation level of VE cadherin in.Pn infection group and bradykinin treatment group was significantly higher than that in normal control group, the difference was statistically significant (P0.05); the tyrosine phosphorylation level of VE cadherin Y658 loci in C.pn infection + chloroquine treatment group was significantly higher than that of C.pn infection group, and the difference has the significance of overall planning (P0.05). Conclusion: C.pn infection may be promoted by V. Phosphorylation of E cadherin Y658 site promotes VE endocytosis and increases VEC permeability.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R543.5

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