本文選題：組織蛋白酶L + 氧化低密度脂蛋白��； 參考：《南華大學(xué)》2012年碩士論文

【摘要】：【目的】 觀察組織蛋白酶L（cathepsin L,CATL）在氧化低密度脂蛋白（oxidized lowdensity lipoprotein, Ox-LDL）誘導(dǎo)單層臍靜脈內(nèi)皮細胞（HUVECs）通透性增加中的作用及其機制探討。 【方法】 （1）用不同濃度Ox-LDL處理HUVECs24h，Transwell和熒光分光光度法檢測HUVECs通透性變化；逆轉(zhuǎn)錄聚合酶鏈反應(yīng)（RT-PCR）和免疫印記技術(shù)（Western blot）分別檢測HUVECs CATL、TET2、VE-cadherin、自噬和凋亡指標(biāo)的mRNA及蛋白表達情況；用CATL活性檢測試劑盒檢測HUVECs CATL活性水平；MDC染色觀測HUVECs自噬水平，Hochest染色和流式細胞術(shù)檢測HUVECs凋亡水平。 （2）用不同濃度CATL inhibitor預(yù)孵育HUVECs24h，再加入50mg/LOx-LDL處理24h，檢測指標(biāo)同前（1）。 （3）用TET2siRNA轉(zhuǎn)染HUVECs，Western blot檢測Beclin-1、LC3、Caspase-3和VE-cadherin蛋白表達。 【結(jié)果】 （1）用Ox-LDL（0、25、50和75mg/L）處理HUVECs，實驗結(jié)果顯示：隨著Ox-LDL濃度的增加，CATL mRNA表達差異無統(tǒng)計學(xué)意義（P0.05），，但其蛋白表達及活性顯著增加（P0.01）；VE-cadherin蛋白表達減少(P0.05)，單層HUVECs通透性增加(P0.05)。Beclin-1mRNA和蛋白表達明顯增加，LC3mRNA表達差異無顯著性，但LC3Ⅰ/LC3Ⅱ明顯增加；MDC染色結(jié)果顯示自噬囊泡染色增加；Caspase-3mRNA表達在50mg/L Ox-LDL組顯著上調(diào)（P0.001）；Ox-LDL處理組Caspase-3蛋白表達顯著高于對照組（P0.05）；Caspase-9mRNA表達差異無統(tǒng)計學(xué)意義（P0.05）；Bcl-2mRNA表達呈Ox-LDL處理濃度依賴性降低（P0.01）；Hochest染色和流式細胞術(shù)結(jié)果顯示Ox-LDL促進HUVECs凋亡。 （2）用不同濃度CATL inhibitor預(yù)孵育HUVECs24h，再加入50mg/LOx-LDL處理24h，實驗結(jié)果顯示：隨著CATL inhibitor預(yù)處理濃度的增加，Ox-LDL誘導(dǎo)的單層HUVECs通透性降低（P0.01）；VE-Cadherin表達增加（P0.01）；Beclin-1和LC3mRNA表達差異不顯著（P0.05），Beclin-1蛋白表達及LC3Ⅰ/LC3Ⅱ降低（P0.01）；MDC染色結(jié)果顯示自噬囊泡減少；Caspase-3mRNA和蛋白表達上調(diào)（P0.05），Caspase-9mRNA表達差異無顯著性（P0.05）； Bcl-2mRNA表達增加（P0.01）；Hoechst染色和流式細胞術(shù)結(jié)果顯示CATL inhibitor上調(diào)Ox-LDL誘導(dǎo)的HUVECs凋亡。 （3）Ox-LDL呈劑量依賴性下調(diào)TET2mRNA和蛋白表達（P0.05），CATLinhibitor呈劑量依賴性上調(diào)Ox-LDL處理的HUVECs TET2mRNA和蛋白表達。TET2siRNA顯著上調(diào)HUVECs Beclin-1蛋白表達（P0.001）和LC3Ⅱ/LC3Ⅰ（P0.01）， TET2siRNA下調(diào)HUVECs Caspase-3蛋白表達（P0.01）；但TET2siRNA對HUVECs VE-Cadherin蛋白表達差異無統(tǒng)計學(xué)意義（P0.05）。 【結(jié)論】 CATL介導(dǎo)Ox-LDL對血管內(nèi)皮細胞自噬和凋亡的調(diào)節(jié)以及內(nèi)皮細胞單層通透性的改變，TET2參與此過程。
[Abstract]:[objective] to investigate the effect of cathepsin L (cathepsin L) on the permeability of monolayer umbilical vein endothelial cells (HUVECs) induced by oxidized low density lipoprotein (oxidized lowdensity lipoprotein,) and its mechanism. [methods] (1) HUVECs were treated with different concentrations of Ox-LDL for 24 h and the permeability of HUVECs was detected by fluorescence spectrophotometry. Reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot were used to detect the mRNA and protein expression of HUVECs, autophagy and apoptosis, and the activity of HUVECs CATL was detected by CATTL assay kit. (2) HUVECs were preincubated with different concentrations of CATL inhibitor for 24 h, then treated with 50 mg / L Ox-LDL for 24 h. The detection indexes were detected by Tet 2siRNA transfected HUVECs blot with 1). (3 for 24 h. [results] (1) HUVECs were treated with Ox-LDL (0 ~ 2550 mg / L and 75 mg / L). The results showed that there was no significant difference in the expression of CATL mRNA with the increase of Ox-LDL concentration (P0.05), but its protein expression and activity increased significantly (P0.01). The expression of VE-cadherin protein decreased (P0.05), the permeability of monolayer HUVECs increased (P0.05) .Beclin-1 mRNA and protein expression increased significantly, but LC3 鈪

本文編號：2065702