拟缺血性损伤对rBMECs上P-gp的影响及其机制研究

发布时间：2018-12-11 04:25

【摘要】：目的：探讨拟缺血性损伤对大鼠脑微血管内皮细胞（Rat Brain MicrovascularEndothelial Cells，rBMECs）上P-糖蛋白（P-glycoprotein，P-gp）的影响及其相关机制。方法：原代培养rBMECs、神经元和神经胶质细胞并借助Transwell建立共培养体系；在共培养体系的基础上用Na2S2O4（终浓度为2mM）建立损伤模型,损伤时间为4h。在共培养体系内室、外室加入肿瘤坏死因子（Tumor necrosis factor-α，TNF-α）、一氧化氮合酶（Nitric-oxide synthase，NOS）、蛋白激酶C（Protein kinase C，PKC）的抑制剂H398、L-NMMA(NG-Monomethyl-L-arginine.monoacetate）、BIM（Bisindolylmaleimide)测定Rh123的表观渗透系数（Apparent Permeability Coefficient，Papp）以反映拟缺血性损伤对rBMECs上P-gp功能的变化；测定外室荧光素钠（Fluorescein Sodium Injection，NaF）的Papp以确保rBMECs紧密连结的完整性；采用Western Blot法确定可能引起P-gp表达和功能变化的因子，然后加入相应的抑制剂测定各因子和P-gp表达变化的情况，研究拟缺血性损伤对rBMECs上P-gp的影响的机制。结果：1.通过Transwell成功建立了rBMECs、神经元和神经胶质细胞的共培养体系。2.以Na2S2O4（终浓度为2mM）诱导rBMECs损伤4h，与空白组比较，损伤组Rh123由内室到外室的Papp值显著降低，，由外室到内室的Papp值显著增强，说明P-gp的功能是增强的；NaF的Papp值无显著变化表明rBMECs紧密连结没有受到影响，确保了实验结果的准确性。3.Western Blot结果显示，Na2S2O4（终浓度为2mM）诱导rBMECs损伤4h，TNF-α、NOS和PKC表达均明显增强；H398对TNF-α表达无明显影响但可下调NOS、PKC、P-gp的表达；L-NMMA对TNF-α表达无明显影响但可下调、PKC、P-gp的表达；BIM对TNF-α、NOS的表达无明显影响但可下调P-gp的表达。结论：拟缺血性损伤时，rBMECs受到刺激合成并释放促炎症细胞因子TNF-α，TNF-α继而激活NOS、PKC对rBMECs上P-gp起调控作用且上调其表达，为脑中风药物治疗的研究提供了新的特异性靶点。
[Abstract]:Aim: to investigate the effect of ischemic injury on P-glycoprotein (P-glycoprotein P GP) in rat brain microvascular endothelial cells (Rat Brain MicrovascularEndothelial Cells,rBMECs) and its related mechanism. Methods: rBMECs, neurons and glial cells were cultured in primary culture and co-culture system was established by means of Transwell. On the basis of co-culture system, Na2S2O4 (final concentration was 2mM) was used to establish the injury model for 4 h. Tumor necrosis factor (Tumor necrosis factor- 伪 (TNF- 伪), nitric oxide synthase (Nitric-oxide synthase,NOS), protein kinase C (Protein kinase (C (Protein kinase) inhibitor H398 were added into the external chamber of co-culture system. L-NMMA (apparent osmotic coefficient (Apparent Permeability Coefficient,Papp) of Rh123 was determined by NG-Monomethyl-L-arginine.monoacetate), BIM (Bisindolylmaleimide) to reflect the changes of P-gp function on rBMECs induced by pseudo ischemic injury. The Papp of sodium fluorescein (Fluorescein Sodium Injection,NaF) was determined to ensure the integrity of rBMECs. Western Blot method was used to determine the factors that might cause the changes of P-gp expression and function, and then the corresponding inhibitors were added to determine the changes of factors and P-gp expression, to study the mechanism of the effect of pseudo ischemic injury on P-gp on rBMECs. Results: 1. The co-culture system of rBMECs, neurons and glial cells was successfully established by Transwell. 2. RBMECs injury induced by Na2S2O4 (final concentration 2mM) was induced for 4 h. Compared with the blank group, the Papp value of Rh123 in the injured group decreased significantly from the inner chamber to the outer chamber, and the Papp value from the outer chamber to the inner chamber was significantly enhanced, which indicated that the function of P-gp was enhanced. There was no significant change in the Papp value of NaF, which indicated that the close connection of rBMECs was not affected, and the accuracy of the experimental results was ensured. 3.Western Blot results showed that the expression of TNF- 伪, NOS and PKC in rBMECs injury induced by Na2S2O4 (final concentration was 2mM) was significantly increased. H398 had no obvious effect on the expression of TNF- 伪 but could down-regulate the expression of NOS,PKC,P-gp, L-NMMA had no obvious effect on the expression of TNF- 伪, but could down-regulate the expression of PKC,P-gp. BIM had no significant effect on the expression of TNF- 伪 and NOS, but could down-regulate the expression of P-gp. Conclusion: during ischemic injury, rBMECs is stimulated to synthesize and release pro-inflammatory cytokines TNF- 伪, TNF- 伪 and then activate NOS,PKC to regulate and up-regulate the expression of P-gp on rBMECs. It provides a new specific target for the drug therapy of cerebral apoplexy.
【学位授予单位】：河南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R743

【参考文献】