【作者】 丁艳芳；

【导师】 杨佩满；

【作者基本信息】 大连医科大学 ， 人体解剖与组织胚胎学， 2003， 硕士

【摘要】 目的：多药耐药性（multidrug resistance，MDR）是指肿瘤细胞对一种抗肿瘤药物产生抗药性的同时，对结构及作用机制不同的多种抗肿瘤药亦产生耐药的现象，是目前化疗失败的主要原因之一。MDR的机制复杂多样，一种肿瘤耐药细胞往往涉及多种耐药机制，导致单一用药逆转MDR效果不理想。因而联合应用两种逆转剂，特别是两种中药，可能针对不同的机制而取得更好的化疗效果，同时具有毒副作用小的优点。已有研究表明，三氧化二砷及苦参碱单独应用均能不同程度的逆转MDR，但有关三氧化二砷及苦参碱联合逆转肿瘤细胞MDR的研究尚未见报道。本研究应用三氧化二砷及苦参碱联合逆转有多药耐药表型的人白血病细胞株K562／ADM对阿霉素的耐药性，观察联合用药逆转耐药的疗效，并从P-糖蛋白介导的药物转运能力的改变及细胞内药物代谢相关物质解毒功能的改变两方面探讨其逆转机制，旨在为临床逆转MDR提供新的策略及理论依据。 方法：实验组为下列几组：（1）．K562／S敏感细胞组 （2）．K562／ADM耐药细胞组 （3）．K562／ADM+As₂O₃组 （4）．K562／ADM+苦参碱组 （5）．K562／ADM+As₂O₃+苦参碱组。MTT法测定逆转剂三氧化二砷或／和苦参碱的细胞毒性及药物作用后的逆转倍数，荧光分光光度法检测细胞内药物（ADM）浓度的改变，流式细胞术测定细胞凋亡百分率的变化，以评价三氧化二砷、苦参碱单独及联合与阿霉素共同作用情况下对K562／ADM阿霉素耐药的逆转效果。免疫组化法检测mdr-1基因编码的P-gp和GST-π基因编码的GST-π表达变化，并用计算机图象分析系统进行半定量分析，流式细胞术定量检测P-gp变化，生化法测定细胞内GSTs酶活性变化，以探讨三氧化二砷及苦参碱逆转MDR机制与P-gp介导的药物转运能力改变及细胞内药物代谢相关物质解毒功能改变之间的关系。 结果：1．三氧化二砷和苦参碱对K562／S及K562／ADM均有明显的抑制作用，且IC₅₀接近，无统计学差异（P＞0.05）。并分别确定了三氧化二砷及苦参碱对K562／ADM细胞的非细胞毒性剂量和低细胞毒性剂量，并以前者作为最佳的逆转浓度，同时还确定了非细胞毒性剂量的两药联合应用后未出现毒性叠加。2.在与ADM合用时，K562/ADM+AsZO。组及K562/A DM+苦参碱组的ICS。均低于K562/ADM耐药组（P<0.01），但仍高于K562/ADM+AsZO3+苦参碱组（P<0.01），二药联合应用时逆转倍数大于两者单独作用之和。K562/ADM+As20。组及K562/ADM+苦参碱组，细胞内化疗药物ADM浓度和细胞凋亡百分率均高于 K562/ADM耐药组（P<0.05，P<0.01），但均低于K562/ADM+As203+苦参碱组（P<0.01，P<0.05）。3.免疫组化法结果显示，与K562/S比较，K562/ADM细胞中P一gP及GST一:呈高表达，且生化法显示细胞中的GSTs酶活性增强。4.与K562/ADM耐药组比较，K562/ADM+ASZO3组细胞内GST一:含量及细胞中的GSTs酶活性均降低（P<0.01），而K562/ADM+苦参碱组无明显变化（P>0.05）。5.与K562/ADM耐药组比较，K562/ADM+苦参碱组P一gP表达量降低（P<O‘01），而K562/ADM+AsZO3组P一gP表达量无明显变化（P>0.05），但当两者联合应用时作用大于两者单独作用之和。 结论:1.三氧化二砷和苦参碱均是有效的抗肿瘤药，且K562/ADM对其不具耐药性，非细胞毒性剂量的两药联合时无毒性的叠加。2.非细胞毒性剂量的三氧化二砷和苦参碱均可部分逆转有多药耐药表型的细胞株K562/ADM对阿霉素的耐药J险，二者联合应用效果优于单独应用，具有协同作用。3.K562/ADM细胞中多药耐药基因mdr一1编码的P一gP和GST一:基因编码的GST一:的过度表达，以及细胞内GSTS酶活性的增强可能是引起K562/ADM细胞产生MDR的主要原因。4.三氧化二砷与苦参碱联合用于逆转MDR时，针对不同的机制，三氧化二砷主要通过下调GST一:表达及抑制GSTs酶活性，而苦参碱主要通过下调P一gP的表达。更多还原

【Abstract】 Objective: multidrug resistance (MDR) means that tumor cells produce the resistance to multiple chemotherapy agents, which is the major cause of failure of chemotherapy. The mechanism of MDR are complex and variable. One kind of drug resistance cell may concern several mechanism of MDR, leading to unsatisfying effect with single reversal agent. If two reversal agents, especially two kind of Chinese medicine, combined together aiming at different mechanism , better chemotherapy effect maybe can obtained with less toxicity. It has been identified that both As2O3 and matrine have reversal effect on tumor cells. But the research about combination of As2O3 and matrine to reverse MDR of tumor cells has not been reported at present. This study was planned to observe the reversal effect of combination of AS2O3 and matrine on K562/ADM cells, a adriamycin-resistant cell line of human leukemia. The relationship between their reversal effect with changes of cellular drug efflux mediated by P-gp and changes of GST- π related to drug detoxification was also studied, aiming to find effective strategy and its theoretical basis for reversing MDR in clinic.Methods: The major group were divided as fllows: (1).K562/S cell line. (2). K562/ADM cell line. (3). K562/ADM cell line with As2O3.(4). K562/ADM cell line with matrine.(5). K562/ADM cell line with As2O3 and matrine . Cell toxicity and reversal times of As2O3 and/or matrine on tumor cells were determined by MTT assay. Intracellular drug concentration and cell apoptosis percentage were determined by spectrophotofluorometry and flow cytometry respectively. The above-mentioned index were used to value the reversal effect of As2Oj and matrine along with ADM on K562/ADM singlely or combined used. In order to discuss the mechanism of reversal effect of As2O3 and matrine, the following index were measured: P-gp coded by mdr-1 gene and GST- n expression were observed via immunohistochemical technique andsemi-quantified by computer image analysis system; changes of P-gp were determined by flow cytometry and changes of GSTs enzymic activity by biochemical method.Results: 1. As2O3 or matrine could obviously inhibit the growth of K562/S and K562/ADM, its’ IC50 had not statistical difference (P>0.05).Non-cytotoxic dosage and low-cytotoxic dosage of As2O3 and matrine were determined by MTT assay .Non-cytotoxic dosage was selected as reversal dosage. 2.Used along with ADM, IC50 of both K562/ADM with As2O3 and K562/ADM with matrine were lower than that of K562/ADM(P < 0.01),but still higher than that of K562/ADM with As2O3 and martrine(P<0.01). Two agents combined together, the reversal effect was better than that of the sum of As2O3 or matrine singely. Intracellular ADM concentration and cell apoptosis percentage of K562/ADM with As2O3 or matrine was higher than that of K562/ADM (P<0.05,P<0.01,respectively), but still lower than that of K562/ADM with As2O3 and matrine(P<0.01,P<0.05,respectively) S.Immunohistochemical results showed that P-gp and GST- n overexpress in K562/ADM.GSTs enzymic activity were enhanced.at the same time.4.Compared with K562/ADM,intracellular GST- n content and GSTs enzymic activity were lower in K562/ADM with As2O3 (P<0.01). No significant changes were found in K562/ADM with matrine.( P>0.05).5. P-gp expression in K562/ADM with matrine was lower than that of K562/ADM(P< 0.01),but still higher than that of K562/ADM with As2O3 and matrine(P<0.01). There was no significant changes of P-gp expression in K562/ADM with As2O3 compared with K562/ADM(P>0.05).Conclusions: 1. As2O3 and matrine were effective antitumor drugs with obvious inhibiting effect on tumor cells. K562/ADM had no drug resistance to As2O3 and matrine.2.Non cytotoxic dosage of As2O3 and matrine could partly reverse the MDR of K562/ADM. The effect of combination of two agents were superior to that of single uses due to their coordinate effect.3.Overexpression of P-gp and GST- π and enhanced enzymic activity of GSTs were related to mechanism of MDRAThe reversal mechanisms of As2O3 and mat更多还原