CAJ | 학술논문

目的：：探讨脂多糖(LPS)诱导微囊胞吞血管内皮钙黏蛋白(VE-Cad)的可能机制。方法：培养人血管内皮细胞株CRL-2922,当其生长至融合状态时分为正常对照组(不予再处理)、LPS处理组(采用10μg/ml LPS分别与CRL-2922再培养1h、2h、4h和6h)、LPS+抑制剂组包括Toll样受体4(TLR4)抑制剂 CLI-095和 Src 抑制剂SU6566[在 LPS培养细胞时分别加入CLI-095(5μg/ml)和 SU6566(2μmol/L)再培养4h]。采用 Western Blotting法检测各组 Src蛋白表达、Cav1磷酸化和 VE-Cad质膜蛋白表达,以及Cav1与 VE-Cad共沉淀水平；采用培养小室半透膜培养细胞,并检测相关各组细胞荧光透过率,以反映血管通透性。结果：LPS处理不同时间组 Src 蛋白表达均较正常对照组升高(P<0.05)；与正常对照组比较,LPS处理4h组Cav1磷酸化增强(P<0.05)、VE-Cad质膜蛋白表达下调、Cav1与 VE-Cad共沉淀水平升高(P<0.05),单层细胞荧光透光率增加(P<0.05)；TLR4抑制剂和Src抑制剂可显著降低 LPS增高的 Src蛋白高表达和Cav1高度磷酸化(P<0.05),上调 VE-Cad质膜蛋白表达(P<0.05),下调Cav1与 VE-Cad共沉淀水平(P<0.05),改善单层内皮细胞通透性(P<0.05)。结论：LPS可能通过TLR4-Src信号途径诱导微囊胞吞 VE-Cad和增加血管通透性。
목적：：탐토지다당(LPS)유도미낭포탄혈관내피개점단백(VE-Cad)적가능궤제。방법：배양인혈관내피세포주CRL-2922,당기생장지융합상태시분위정상대조조(불여재처리)、LPS처리조(채용10μg/ml LPS분별여CRL-2922재배양1h、2h、4h화6h)、LPS+억제제조포괄Toll양수체4(TLR4)억제제 CLI-095화 Src 억제제SU6566[재 LPS배양세포시분별가입CLI-095(5μg/ml)화 SU6566(2μmol/L)재배양4h]。채용 Western Blotting법검측각조 Src단백표체、Cav1린산화화 VE-Cad질막단백표체,이급Cav1여 VE-Cad공침정수평；채용배양소실반투막배양세포,병검측상관각조세포형광투과솔,이반영혈관통투성。결과：LPS처리불동시간조 Src 단백표체균교정상대조조승고(P<0.05)；여정상대조조비교,LPS처리4h조Cav1린산화증강(P<0.05)、VE-Cad질막단백표체하조、Cav1여 VE-Cad공침정수평승고(P<0.05),단층세포형광투광솔증가(P<0.05)；TLR4억제제화Src억제제가현저강저 LPS증고적 Src단백고표체화Cav1고도린산화(P<0.05),상조 VE-Cad질막단백표체(P<0.05),하조Cav1여 VE-Cad공침정수평(P<0.05),개선단층내피세포통투성(P<0.05)。결론：LPS가능통과TLR4-Src신호도경유도미낭포탄 VE-Cad화증가혈관통투성。
Objective:To observe the mechanism of caveolae-mediated endocytosis of VE-Cad after LPS treat-ment.Method:Human vascular endothelial cell line CRL-2922 was cultured.It was divided into normal control group (when it grew to confluence state),LPS-treated group (using 10μg/ml LPS to incubate with the CRL-2922 for 1h,2h,4h and 6h);and LPS+inhibitor group[adding CLI-095 (5μg/ml)and SU6566 (2μmol/L)in the LPS cultured cells and then culturing for 4h].Western Blotting was used to detect the Src protein expression,Cav1 phosphorylation,plasma membrane protein expression of VE-Cad,phosphorylation of Cav1 and co-precipitation lev-els of Cav1 and VE-Cad;semi-permeable membrane was used to culture cells,and the relevant cells fluorescence transmittance was detected to reflect vascular permeability.Results:The protein expression of Src was gradually in-creased after LPS treatment,as well as the phosphorylation (Tyr14)of Cav1 and he monolayer cell permeability (P<0.05),and the membrane expression of VE-Cad decreased (P<0.05).The increased expression and phosphorylation could be decreased by the inhibitor of TLR4 CLI-095 and the inhibitor of Src SU6656 (P<0.05),and the inhibitors could increase the membrane expression of VE-Cad (P<0.05),and improve the monolayer cell permeability at 4h after LPS treatment (P<0.05).Conclusion:Caveolae-mediated endocytosis of VE-Cad was activated through LPS-TLR4-Src signal pathway.