本文選題：肺炎衣原體 + 血管內(nèi)皮細胞�。� 參考：《天津醫(yī)科大學(xué)》2017年碩士論文

【摘要】：利用肺炎衣原體(Chlamydia pneumoniae,C.pn)體外感染人臍靜脈內(nèi)皮細胞系EA.hy926細胞模型,觀察C.pn感染對血管內(nèi)皮細胞(Vascular endothelial cell,VEC)通透性的影響,并從血管內(nèi)皮細胞鈣粘素(Vascular endothelial cadherin,VE cadherin)內(nèi)吞的角度探討C.pn感染增加VEC通透性的機制,證實C.pn感染可通過促使VE鈣粘素磷酸化促進VE鈣粘素內(nèi)吞而增加VEC通透性。方法:1.免疫熒光法檢測VEC生長致密時的細胞間連接;2.測量累計熒光透過率檢測C.pn感染對VEC通透性的影響;3.CCK-8實驗檢測氯喹在作用時間和工作濃度內(nèi)對VEC的毒性;4.用氯喹處理VEC后,采用免疫熒光法觀察C.pn感染后對VE鈣粘素內(nèi)吞的影響;5.CCK-8實驗檢測血管內(nèi)皮生長因子(Vascular endothelial growth factor,VEGF)在作用時間和工作濃度內(nèi)對VEC的毒性;6.應(yīng)用Western blot實驗檢測C.pn感染VEC后VE鈣粘素總蛋白表達水平的變化;7.利用質(zhì)膜提取試劑盒提取VEC細胞膜蛋白或以胰酶去除細胞膜后提取VEC胞漿蛋白,應(yīng)用Western blot實驗觀察C.pn感染后VE鈣粘素的內(nèi)吞情況;8.CCK-8實驗檢測內(nèi)吞抑制劑氯丙嗪在作用時間和工作濃度內(nèi)對VEC的毒性;9.測量累計熒光透過率檢測加入氯丙嗪后,C.pn感染對VEC通透性的影響;10.CCK-8實驗檢測緩激肽在作用時間和濃度內(nèi)對VEC的毒性;11.應(yīng)用Western blot實驗檢測Src激酶抑制劑PP2對C.pn感染促進VE鈣粘素Y658位點磷酸化的影響;12.測量累計熒光透過率觀察PP2對C.pn感染增加VEC通透性的影響;13.利用質(zhì)膜提取試劑盒提取VEC細胞膜蛋白或以胰酶去除細胞膜后提取VEC胞漿蛋白,應(yīng)用Western blot實驗檢測PP2對C.pn感染促進VE鈣粘素內(nèi)吞的影響。結(jié)果:1.光學(xué)顯微鏡下可見,生長致密的VEC細胞間連接完整;2.C.pn感染VEC 0 h、18 h、24 h、36 h、48 h后,VEC通透性明顯增高,其中以感染24 h后增高最為明顯,差異具有統(tǒng)計學(xué)意義(P0.05);3.CCK-8實驗結(jié)果顯示,以20μmol/l、40μmol/l、60μmol/l和80μmol/l工作濃度的氯喹處理VEC 24 h對VEC活力無明顯影響,差異無統(tǒng)計學(xué)意義,而以100μmol/l的氯喹處理VEC 24 h后,VEC活力明顯下降,差異有統(tǒng)計學(xué)意義(P0.05);4.激光共聚焦結(jié)果顯示,正常VEC內(nèi)呈特征性熒光綠色的VE鈣粘素均勻分布于細胞與細胞連接處。C.pn感染24 h后,VE鈣粘素在VEC胞膜處表達明顯減少,但在胞漿中表達增多,且呈彌散分布,細胞間連接不再清晰分明,并出現(xiàn)明顯縫隙。而氯喹處理后,C.pn感染引起的VE鈣粘素由胞膜向胞漿內(nèi)分布即VE鈣粘素內(nèi)吞更為明顯。5.CCK-8實驗結(jié)果顯示,在工作時間內(nèi),工作濃度的VEGF對VEC活力無明顯影響,差異無統(tǒng)計學(xué)意義(P0.05);6.Western blot結(jié)果顯示:C.pn感染組、VEGF組、C.pn感染+氯喹處理組、VEGF+氯喹處理組以及氯喹處理組與正常對照組相比,VE鈣粘素總蛋白表達水平均無明顯變化(P0.05);7.Western blot結(jié)果顯示:C.pn感染組和VEGF處理組的細胞膜VE鈣粘素表達水平與正常對照組明顯減少,差異具有統(tǒng)計學(xué)意義(P0.05),而正常對照組細胞漿中僅能檢測到少量的VE鈣粘素,C.pn感染組與VEGF處理組的細胞漿中VE鈣粘素較正常對照組有所增加;應(yīng)用溶酶體抑制劑氯喹抑制內(nèi)吞的VE鈣粘素降解后,C.pn感染+氯喹處理組與C.pn感染組相比,其細胞膜上VE鈣粘素表達水平無明顯差異,但前者細胞漿中VE鈣粘素表達水平較后者有所增加,差異具有統(tǒng)計學(xué)意義(P0.05);VEGF+氯喹處理組與VEGF處理組的VEC細胞膜上VE鈣粘素表達水平無明顯增加,但與后者相比,前者細胞漿中VE鈣粘素表達水平明顯增加,差異具有統(tǒng)計學(xué)意義(P0.05),提示C.pn感染可明顯促進VE鈣粘素內(nèi)吞。8.CCK-8實驗結(jié)果顯示,在工作時間內(nèi),工作濃度的氯丙嗪對VEC活力無明顯影響,差異無統(tǒng)計學(xué)意義(P0.05);9.通過測量累計熒光透過率發(fā)現(xiàn),加入氯丙嗪抑制VE鈣粘素內(nèi)吞后,C.pn感染引起的VEC通透性增加被明顯抑制(P0.05);10.CCK-8實驗結(jié)果顯示,在工作時間內(nèi),工作濃度的緩激肽對VEC活力無明顯影響,差異無統(tǒng)計學(xué)意義(P0.05);11.Western blot結(jié)果顯示:C.pn感染組和緩激肽處理組VE鈣粘素磷酸化水平較正常對照組顯著增加,差異有統(tǒng)計學(xué)意義(P0.05);PP2+C.pn感染組的VE鈣粘素磷酸化水平較C.pn感染組明顯下降,差異有統(tǒng)計學(xué)意義(P0.05);12.累計熒光透過率檢測結(jié)果顯示,PP2預(yù)處理可削弱緩激肽促進VEC通透性增加的作用,差異具有統(tǒng)計學(xué)意義(P0.05);同時,PP2預(yù)處理VEC后,C.pn感染增加VEC通透性的作用也明顯減弱,差異具有統(tǒng)計學(xué)意義(P0.05),提示C.pn感染可通過促進VE鈣粘素磷酸化而增加VEC通透性;13.Western blot結(jié)果顯示,C.pn感染組和緩激肽處理組的細胞膜VE鈣粘素磷酸化水平與與正常對照組相比明顯增加,差異有統(tǒng)計學(xué)意義(P0.05);而PP2+C.pn感染組的VE鈣粘素磷酸化水平較C.pn感染組明顯減少,差異有統(tǒng)計學(xué)意義(P0.05);且在細胞漿中,C.pn感染組和緩激肽處理組的VE鈣粘素磷酸化水平較正常對照組明顯增加,差異有統(tǒng)計學(xué)意義(P0.05);C.pn感染+氯喹處理組的VE鈣粘素Y658位點的酪氨酸磷酸化水平較C.pn感染組明顯增加,差異有統(tǒng)計學(xué)意義(P0.05)。結(jié)論:C.pn感染可能通過促進VE鈣粘素Y658位點的磷酸化促進VE鈣粘素內(nèi)吞,進而增加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.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R543.5

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本文編號：1964846