难治性癫痫患者脑组织中耐药蛋白表达与卡马西平浓度的相关性

The relationship between expression of drug resistance proteins and antiepileptic drug concentrations in brain tissue of patients with refractory epilepsy

目的通过对难治性癫痫患者脑组织中P-糖蛋白、肺耐药相关蛋白表达与卡马西平浓度的比较及相关性研究，探讨难治性癫痫耐药的机制。方法选取2007年至2009年间于宣武医院功能神经外科行癫痫致痫灶切除术治疗且术前服用卡马西平半年以上的26例药物难治性癫痫患者的脑组织标本（其中局灶性皮质发育不良Ⅰb10例、Ⅱa4例、Ⅱb2例，神经节细胞胶质瘤6例，胚胎发育不良性神经上皮瘤4例）。应用Envision二步法进行免疫组织化学标记，观察两种耐药蛋白在脑组织中的表达部位和表达强度。应用Western blot法进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳，对两种耐药蛋白在脑组织病灶区域和病灶周围区域的表达分别进行定量分析。应用荧光偏振免疫分析法对病灶区域和病灶周围区域的卡马西平浓度进行测定。结果 P-糖蛋白在局灶性皮质发育不良Ⅱ型和难治性癫痫相关脑肿瘤病例的病灶区域表达（μg/m1）均高于病灶周围区域（分别是2．593±0．829和1．711±0．292，t=-2．201，P=0．028；1．352±0．445和1．179±0．593，t=2．698，P=0．028）。肺耐药相关蛋白在局灶性皮质发育不良Ⅱ型和难治性癫痫相关脑肿瘤病例的病灶区域表达（μg／m1）亦均高于病灶周围区域（分别是1．567±0．092和0．775±0．101，t=-2．516，P=0．024；1．091±0．239和0．825土0．297，t=3．997，P=0．003）。卡马西平在难治性癫痫相关脑肿瘤病灶区域平均浓度（μg/m1）低于病灶周围区域（0．848±0．726和0．948土0．785，t=-3．056，P=0．014），在病灶区域卡马西平浓度低于病灶周围区域的8例中，病灶区域P-糖蛋白和肺耐药相关蛋白同时升高。但是P-糖蛋白、肺耐药相关蛋白表达量与难治性癫痫患者脑组织卡马西平浓度无显著的等级相关性。结论耐药蛋白参与了难治性癫痫耐药的过程，同时提示癫痫的耐药是一个复杂的过程，不同病变可能有不同的机制参与了耐药过程。

Objective To compare the expression and distribution of drug resistance proteins P- glycoprotein （P-gp） and lung resistance protein （LRP） in brain tissues of patients with refractory epilepsy and to investigate the relationship between expression of drug resistance proteins and concentration of antiepileptie drug carbamazepine （CBZ）. Methods We included the brain tissues of 26 eases with refractory epilepsy who had in the experiment. They had following pathologic diagnosis： focal cortical dysplasia （FCD）Ⅰ b （ n = 10）, FCD Ⅱ a （ n = 4）, FCD Ⅱb （ n = 2 ） and brain tumorsincluding ganglioglioma （n =6） and dysembryoplastic neuroepithelial tumor（n =4）. Immunohistochemistry staining using EnVision system was used to reveal the expression location of P-gp and LRP, and Western blot in SDS-polyacrylamide gel was used to quantitatively analyze the expression of P-gp and LRP. Fluorescence polarization immunoassay was used to determine concentration of CBZ. Results Both P-gp and LRP performed （μg/ml） prominent overexpression in brain tissues of patients with refractory epilepsy, especially in the lesions of both FCD type Ⅱ （P-gp ： 2.593 ±0.829 vs 1.711 ±0.292, t = -2.201,P=0.028;LRP：1. 352±0. 445 vs 1. 179 ±0. 593, t = -2. 516,P =0. 028, respectively）and tumor（P-gp：l. 567 ±0. 092 vs 0.775±0.101, t =2.698, P =0.024;LRP：l.091 ±0.239 vs 0.825 ±0.297,t =3.997,P =0.003 respectively）. The concentration of CBZ in lesions of brain tumors were lower than which in surrounding regions（0. 848±0. 726 vs 0. 948 ±0. 785, t = -3. 056,P =0. 014） , while P-gp and LRP were higher than which in surrounding regions in 80% cases. There was no relationship between expression of drug resistance proteins and conce^ltration of CBZ. Conclusions In processing of drug resistance, P-gp and LRP play important roles. However, there is no correlation between expression of drug resistance proteins and concentration of CBZ, suggesting epilepsy drug resistance to be a complicated mechanism.