人脑胶质瘤细胞多药耐药性的形成及其耐药特性的研究
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【摘要】 目的 探讨人脑胶质瘤细胞多药耐药性(MDR)的形成规律及其耐药特性。方法 采用长春新碱(VCR)在体外对人脑胶质瘤BT325细胞持续诱导获得了表现为MDR特征的人脑胶质瘤细胞模型，以MTT法测定胶质瘤细胞增殖，透射电镜行超微结构研究，通过流式细胞仪，检测培养的胶质瘤细胞与32例术后胶质瘤新鲜组织标本中P-糖蛋白的表达。结果 耐药的胶质瘤细胞BT325／VCR对长春新碱的耐受程度为其亲本细胞的24倍，对阿霉素、威猛交叉耐药，对5-氟脲嘧啶敏感。透射电镜下见BT325／VCR细胞常染色质明显，线粒体丰富，粗面内质网增多 医学教 育网收集整理 。研究表明BT325／VCR表现为P-糖蛋白介导的典型MDR。胶质瘤新鲜组织标本中，P-糖蛋白表达阳性率为37.5%(12／32），同肿瘤的恶性程度呈正相关(P＜0.01)。结论 长春新碱能够诱导人脑胶质瘤细胞产生P-糖蛋白介导的典型MDR。胶质瘤中多药耐药基因产物P-糖蛋白表达的阳性率同肿瘤的恶性程度呈正相关。应用流式细胞仪能早期发现耐药细胞，具有临床推广价值。
Establishment and characterization of multidrug resistance in human g lioma cell line and surgical glioma specimens
FAN Yimin, WANG Hongqin, LI Baoyu, et al.
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Department of Neurosurger y, The First Clinical College of Shanxi Medical University, Tai Yuan 030001
【Abstract】 Objective To try to explain the machanims and characteristics of multidrug resistance in human glioma and to inv e stigate the clinical significance of predicting the responsiveness of individual glioma cells to chemotherapeutic agents.Methods Multidr ug resistant glioma subline was isolated from BT325 human glioma cell line under selection pressure of vincristine. The human glioma cells growth was measured b y MTT assay in vitro. Electronic transmission microscope was used to e xamine the fine str ucture of cultured human glioma cells. The expression of P-glycoprotein in cult u red human glioma cell line and 32 surgical glioma specimens was tested by flow c ytometer with P-glycoprotein monoclonal antibody JSB1.Results The multidrug resistant glioma subline-BT325/VCR was resisted to vincrist i ne, 24 times as high as that of BT325 human glioma cell line, and also significa ntly resisted to adriamycin and teniposide, but sensitived to fluracil. Under e l ectronic transmission microscope, the BT325/VCR subline was documented to be rich in m itochrodria, rough endoplasmic reticulum and euchromatin. The BT325/VCR cell lin e showed a overexpression of P-glycoprotein and P-glycoprotein medicated typic al multidrug resistance phenotype. In 32 surgical glioma specimens, there was no e vidence of complete absense of P-glycoprotein, with a definifion of more than 3 7 .5%(12/32) of P-glycoprotein as positive. Significant positive correlation bet we en the positive rate of P-glycoprotein expression and the degree of tumour mali g nancy were also found.Conclusions Vincristine could ind u ce P-glycoprotein mediated typical multidrug resistance. Multidrug resistance n a turally occurred in many human gliomas, and well correlated between the positive expression rate and the malignancy in glioma. The flow cytometer assay for P-g l ycoprotein detection might be preferable to determinations of multidrug resistan t glioma cells, thereby be beneficial for the design of treatment protocols.
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【Key words】 Glioma Multidrug resistance P-glycoprotein Flow cytometer Vincristine
恶性胶质瘤化疗失败的主要原因之一为肿瘤细胞对化疗药物产生的多药耐药性(Multidrug resistance, MDR)。MDR即对一种化疗药物耐药的肿瘤，同时对另外一些与之化学结构、作用机制完全不同的化疗药物的交叉耐药。典型的MDR是由一种被多药耐药基因(mdrl)编码的膜糖蛋白——P-糖蛋白(P-glycoprotein,P-gp)过度表达所致［1，2］。研究胶质瘤多药耐药细胞形成规律及其耐药特性，有助于深入认识肿瘤MDR机理和寻找新的治疗对策。
材料与方法
1.材料来源：P-糖蛋白单克隆抗体JSB-1购自英国BMB公司，MTT为Sigma产品。人脑多形性胶质母细胞瘤细胞株BT325引自北京市神经外科研究所。全部组织标本选自1996年6月～1997年3月间在我院住院手术的脑胶质瘤患者。
2.实验方法：人脑多形性胶质母细胞瘤BT325细胞株常规无菌培养。采用逐级递增培养液中长春新碱的浓度，使其产生多药耐药［3］。采用改良的MTT法［4］，检测细胞生长及对化疗药物的耐药性。药物诱导过程中，在倒置显微镜下直接观察细胞形态变化。收集BT325及BT325／VCR细胞，固定，包埋，制片，采用透射电镜观察。流式细胞仪检测脑胶质瘤细胞P-糖蛋白表达［5，6］。
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