P糖蛋白在线粒体的定位及功能表达与肿瘤细胞多药耐药相关性研究

Overexpression of the membrane P-glycoprotein (P-gp) is the most important molecular mechanism underlying multidrug resistance(MDR). But the controversial is that the sensitivity of the tumor cells was not fully restored after the addition of P-gp inhibitors. It was found that mitochondria played a key role in the apoptotic process. Some studies implicated that the expression of P-gp participated in the regulation of the activity of the volume sensitivity outwardly rectifying Cl- (VSOR Cl-) channels which were associated with the induction of apoptotic volume decrease(AVD). The AVD is the early event of programmed cell death induced by mitochondria. And the depletion of mitonchondrial DNA was related to increasing P-gp translocation to mitonchondria. Based on this, we want to isolate and purify P-gp from mitochondria from tumor cells to investigate the localization and pump functional characterization of P-gp in mitochondria. Using Patch Clamp, laser capture microdissection and Ciphergen Systems and etc, we want to explore the correlation between expression of mitochondrial P-gp and VSOR Cl- channels and the impact on apoptosis and drug resistance. We want to investigate the subcelluar MDR mechanism of P-gp through the investigation of transmembrane potential of mitochondria and the activity of protein located on the membrane of mitochondria and expect to find the new target to reverse the MDR.

肿瘤细胞膜上P糖蛋白(Pgp)的异常表达是目前肿瘤多药耐药机制的主要学说之一，然而研究发现细胞膜上过表达的Pgp以及Pgp转运功能受抑制后肿瘤细胞并未出现耐药完全逆转；线粒体凋亡途径是传统凋亡途径之一，Pgp表达影响体积调节性氯离子通道活性从而引起凋亡性容积回缩是凋亡的早期标志，线粒体DNA缺失与其Pgp表达具有相关性，由此推测可能与肿瘤耐药有关。基于此本课题设计拟从线粒体膜表面的Pgp分离纯化着手，研究线粒体上Pgp的定位及其表达对多药耐药机制中的"药泵功能"的影响及辅助作用，运用膜片钳、激光捕获、微量蛋白检测系统等方法，探讨线粒体膜上的Pgp对体积敏感性氯离子通道活性的影响与细胞凋亡、肿瘤细胞耐药的相关性，以及线粒体膜电位的改变及定位在线粒体上的耐药蛋白的功能活性，探讨Pgp在亚细胞水平产生MDR的机制，期望寻找到逆转肿瘤细胞MDR的新特异性靶点，为肿瘤的药物治疗提供新的理论依据。

目的:研究P-glycoprotein（P-gp）在卵巢癌细胞株细胞膜和线粒体上的表达及功能，探讨克服肿瘤多药耐药的新策略。方法：MTT法测细胞的耐药性。应用梯度离心法，蔗糖密度梯度分离A2780/Taxol细胞内的线粒体，建立提取线粒体的方法。应用WB方法，分析P-gp蛋白在细胞和线粒体上的表达水平。应用激光共聚焦技术观察P-gp是否在线粒体上定位。分别应用顺铂和紫杉醇处理细胞株，流式细胞术观察线粒体内RHO123的积累和外排情况。结果：顺铂耐药株细胞上没有P-gp表达，在紫杉醇耐药细胞株中顺利检测到P-gp表达。应用MTT法测A2780、A2780/Taxol细胞的耐药性，A2780细胞的IC50=0.3805ng/ul，A2780/Taxol细胞的IC50=2.513ng/ul。透射电镜下观察所提取的线粒体。结论：P-gp在卵巢癌不同细胞株中表达不一致。在紫杉醇耐药细胞株中发挥外排功能，参与卵巢癌的多药耐药。