P-糖蛋白在神经细胞内的降解机制及其临床意义

发布时间：2018-07-17 16:21

【摘要】：P-糖蛋白(P-glycoprotein, P-gp)是一种ATP依赖性的跨膜转运蛋白，可以将多种结构和药效不同的化合物排出细胞，是产生多药耐药的主要原因之一。目前P-糖蛋白在多要耐药方面的研究主要集中在肿瘤及难治性癫痫的发生，其中肿瘤的发生是原癌基因的激活、抑癌基因的抑制、凋亡基因及修复基因的改变等多因素综合作用的结果，而P-糖蛋白的升高是癫痫发展为难治性癫痫的重要生物学基础，，因此，P-糖蛋白在难治性癫痫中的作用机制研究尤为重要。目前关于P-糖蛋白基因调控及相应的信号传导通路成为P-糖蛋白的研究热点，但是P-糖蛋白处于不断的合成与降解的动态平衡，因此，从P-糖蛋白降解的角度揭示P-糖蛋白在多药耐药中的作用机制是一个新思路。目的研究神经细胞内P-糖蛋白的降解途径并探讨P-糖蛋白在神经细胞内降解对难治性癫痫治疗的临床意义。方法 1.腺病毒作为载体，将ABCB1基因转染到U251细胞，建立高表达P-糖蛋白的细胞模型。 2.在已建立的高表达P-糖蛋白的细胞模型上，不同浓度的特异性蛋白酶体抑制剂MG132、溶酶体抑制剂ChlQ处理细胞24h，同时设定时间依赖组。Western blot检测P-糖蛋白水平的变化情况，明确神经细胞内P-糖蛋白的降解途径。结果 1.蛋白酶体抑制剂MG132处理组，P-糖蛋白在蛋白酶体抑制剂处理后表达明显升高，且P-糖蛋白表达随着MG132浓度的升高而升高。P-糖蛋白表达与蛋白酶体抑制剂处理时间无明显相关性，但是蛋白酶体抑制剂处理可以明显增加P-糖蛋白的表达。 2.溶酶体抑制剂ChlQ处理组，P-糖蛋白的表达与对照组相比无明显异常，且不随溶酶体抑制剂浓度及处理时间变化而变化。结论 1.神经细胞内P-糖蛋白的降解与溶酶体途径无关。 2.神经细胞内P-糖蛋白的降解通过泛素-蛋白酶体途径。 3.抑制泛素-蛋白酶体功能可以增加神经细胞内泛素化P-糖蛋白的含量，引起神经细胞内P-糖蛋白积聚。
[Abstract]:P-glycoprotein (P-gp) is an ATP-dependent transmembrane transporter, which can expel many compounds with different structures and effects, and is one of the main reasons for multidrug resistance. At present, the research on multidrug resistance of P- glycoprotein mainly focuses on the occurrence of tumor and refractory epilepsy, in which tumor is activated by proto-oncogene and suppressor gene is inhibited. The change of apoptosis gene and repair gene is the result of many factors, and the increase of P-glycoprotein is an important biological basis for the development of epilepsy into intractable epilepsy, so it is very important to study the mechanism of P- glycoprotein in refractory epilepsy. At present, the regulation of P- glycoprotein gene and the corresponding signal transduction pathway have become the research hotspot of P- glycoprotein, but P- glycoprotein is in the dynamic balance of synthesis and degradation, so, It is a new idea to reveal the action mechanism of P- glycoprotein in multidrug resistance from the point of view of P- glycoprotein degradation. Objective to study the degradation pathway of P-glycoprotein in nerve cells and the clinical significance of P-glycoprotein degradation in the treatment of intractable epilepsy. Method 1. Adenovirus was used as a vector to transfect ABCB1 gene into U251 cells to establish a cell model of high expression of P- glycoprotein. 2. In the established cell model with high expression of P-glycoprotein, the cells were treated with different concentrations of specific proteasome inhibitor MG132 and lysosomal inhibitor ChlQ for 24 h. To identify the degradation pathway of P-glycoprotein in nerve cells. Result 1. The expression of P- glycoprotein in proteasome inhibitor MG132 group was significantly increased after treatment with proteasome inhibitor, and the expression of P- glycoprotein increased with the increase of MG132 concentration. There was no significant correlation between the expression of P- glycoprotein and the treatment time of proteasome inhibitor. However, proteasome inhibitor treatment significantly increased the expression of P-glycoprotein. 2. 2. The expression of P- glycoprotein in the lysosomal inhibitor ChlQ group was not abnormal compared with the control group, and did not change with the concentration of lysosomal inhibitor and the treatment time. Conclusion 1. The degradation of P-glycoprotein in nerve cells was not related to lysosomal pathway. 2. Degradation of P-glycoprotein in nerve cells through the ubiquitin-proteasome pathway. 3. Inhibiting the function of ubiquitin-proteasome can increase the content of ubiquitin-glycoprotein and induce the accumulation of P- glycoprotein in nerve cells.
【学位授予单位】：泰山医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R742.1

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