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100023 北京市2345信箱	世界华人消化杂志 2001年2月15日;9(2):131-134
Email: wcjd@public.bta.net.cn	世界华人消化杂志 ISSN 1009-3079 CN 14-1260/R
http:// www.wjgnet.com	版权归世界胃肠病学杂志社

⊙研究原著⊙

胃癌多药耐药细胞药物积累的异常

程时丹吴云林章永平乔敏敏郭强苏

上海第二医科大学附属瑞金医院消化科上海市 200025
程时丹，女，1969-07-25生，上海市人，汉族. 1993年上海第二医科大学临床医学系毕业，医学硕士.
项目负责人 程时丹，200025，上海第二医科大学附属瑞金医院消化科.
Department of Gastroenterology, Ruijin Hospital, Shanghai Second Medical University, Shanghai 200025, China
Correspondence to Dr. Shi-Dan Cheng, Department of Gastroenterology, Ruijin Hospital, Shanghai Second Medical University,
Shanghai 200025, China
Tel. 0086-21-64370045 Ext. 665241
Received 2000-05-18 Accepted 2000-06-02

Abnormal drug accumulation in multidrug resistant gastric carcinoma cells

Shi-Dan Cheng, Yun-Lin Wu, Yong-Ping Zhang, Min-Min Qiao and Qiang-Su Guo

Abstract

AIM To study the difference in intracellular concentration of drug between multidrug resistant cells and drug sensitive cells
of gastric carcinoma.

METHODS Multidrug resistant gastric carcinoma cell sublines were established by a stepwise increase of concentration of vincristine in media. The uptake of drug was measured by isotope liquid scintillation counter. Laser confocus microscope was used to study the intracellular distribution of daunorubicin and fluorescent intensity of daunorubicin in drug- resistant and drug-sensitive cells after incubated in drug-free media for various time periods.

RESULTS Fluorescent of daunorubicin was mainly contained in the nuclei of both sensitive and resistant cells. The fluorescent intensity of daunorubicin in drug-resistant cells was lower after incubated in drug-free media for various time periods.

CONCLUSION The multidrug resistant gastric cells present a lower rate of drug uptake, and the rate of efflux drug was greater than the sensitive parent cell lines, resulting in the decrease of the intracellular concentration of anticancer drug in multidrug resistant cells, and the cytotoxity of drug.

Subject headings stomach neoplasms; multidrug resistance; accumulation of drug; vincristine; daunorubicin;
antineoplastic agents

Cheng SD, Wu YL, Zhang YP, Qiao MM, Guo QS. Abnormal drug accumulation in multidrug resistant gastric carcinoma cells. 
Shijie Huaren Xiaohua Zazhi, 2001;9(2):131-134

摘要

目的研究胃癌多药耐药细胞与敏感细胞细胞内药物浓度的差异.

方法通过逐渐递增化疗药物长春新碱浓度诱导胃癌细胞株产生多药耐药性. 应用［³H］长春新碱方法测定多药耐药细胞对化疗药物的摄取；利用抗癌药柔红霉素在肿瘤细胞内的自发荧光，用激光共聚焦显微镜观察柔红霉素在细胞内的分布，及撤去柔红霉素后的不同时间点细胞内柔红霉素荧光强度.

结果柔红霉素在细胞内的荧光主要集中在细胞核，在细胞质内亦有较少的荧光显示. 多药耐药细胞内［³H］长春新碱的浓度明显低于亲代细胞；撤去柔红霉素后各个时间点多药耐药细胞内柔红霉素的荧光强度亦均低于亲代细胞.

结论胃癌多药耐药细胞株对细胞毒药物的摄取低于敏感细胞，外排高于敏感细胞，从而导致药物在细胞内的浓度降低，细胞毒作用减弱.

主题词 胃肿瘤；多药耐药；药物积聚；长春新碱；柔红霉素；抗肿瘤药

程时丹, 吴云林, 章永平, 乔敏敏, 郭强苏. 胃癌多药耐药细胞药物积累的异常. 世界华人消化杂志，2001；9(2)：131-134

0 引言

胃癌是我国发病率和死亡率最高的恶性肿瘤之一^［¹^］，早期诊断率低，进展期胃癌占很大比例. 尽管近年来早期诊断水平有所提高，扩大的根治性手术使患者的生存率有了明显的改善，但是化疗仍是绝大多数患者（包括手术后患者）主要的治疗方法^［²^］. 然而大多数患者常常化疗失败，其中一个主要原因是肿瘤产生了多药耐药性^［^3-7^］. 所谓多药耐药（multidrug resistance，MDR）是指肿瘤细胞对结构与作用机制不同的多种药物如长春新碱、柔红霉素和丝裂霉素等产生的交叉耐药现象^［^8-10^］. 多药耐药现象最早是在中国仓鼠的卵巢癌细胞秋水仙素耐药株中发现的^［¹¹^］，在人类血液系统发生的恶性疾病和某些实体瘤如结肠癌、肝癌等均存在着天然耐药^［^12-16^］，胃癌虽属化疗前低表达的恶性肿瘤，但长期化疗增高了获得性表达，MDR同样是限制胃癌化疗疗效发挥^［^17-20^］. 因此，进行胃癌耐药的研究，对提高临床疗效、降低其死亡率具有十分重要的意义.

1 材料和方法

1.1 材料选择人类胃高分化腺癌MKN45细胞、中分化腺癌SGC7901细胞、低分化腺癌MKN28细胞（本实验室传代细胞）作为亲代细胞株，经逐步递增长春新碱浓度的方法诱导亲代细胞产生多药耐药性^［^{21, 22}^］，形成相应的多药耐药细胞亚株MKN45/VCR1.0, SGC7901/VCR1.0, MKN28/VCR0.1，他们对长春新碱的耐药性分别是亲代细胞的7.09倍、6.31倍和8.92倍，并对柔红霉素、丝裂霉素、顺铂、VP16、5-Fu等抗肿瘤药物均具有交叉耐药性.

1.2 方法

1.2.1 胃癌细胞对化疗药物的摄取 采用同位素液闪方法测定^［^23,24^］. 置大小适当的盖玻片于六孔细胞培养板内. 取对数生长期的亲代细胞和长春新碱耐药诱导细胞各1.0mL（细胞数为1×10⁹·L^-1），加培养液2mL. 37℃孵育过夜. 待细胞贴壁生长后，移去孔内培养液，加含［³H］标记的长春新碱（［³H］VCR）（Amershama Life Science公司出品）的培养液1mL（［³H］VCR的浓度为37MBq·L^-1）于每孔. 于15，30，60，90，120min各取2孔，弃去含药培养液，用预冷的PBS洗3次，滴加3g·L^-1胰酶、EDTA消化细胞1.5min，加PBS 1mL制成细胞悬液，真空抽滤至醋酸纤维滤膜上. 800W白炽灯下烘干，装于闪烁瓶中，加闪烁液（PPO 5.5g，POPOP 0.1g，二甲苯667mL，Triton X-100 333mL）1mL，静置过夜后计cpm值. 重复3次.

1.2.2 胃癌细胞对化疗药物的外排 采用激光共聚焦显微镜观测^［^21-23,25-28^］. 置大小适当的盖玻片于六孔细胞培养板内. 取对数生长期的亲代细胞和长春新碱耐药诱导细胞各1.0mL（细胞数为5×10⁸·L^-1），加培养液2mL, 37℃孵育过夜. 细胞贴壁生长后，去上清液，加入含柔红霉素（daunorubicin，DAU）培养液（DAU质量浓度1.0mg·L^-1），37℃培养1h，弃去含DAU培养液，加入1mL促外排培养液（50mmol·L^-1 Tris-HCl（pH 7.6），0.14mol·L^-1 NaCl，5.0mmol·L^-1 KCl，1.0mmol·L^-1 CaCl₂，0.5mmol·L^-1 MgCl₂，2mmol·L^-1谷胱苷肽，1×最小量基础培养液的氨基酸，叠氮钠10mmol·L^-1）37℃孵育15，30，45，60min，激光共聚焦显微镜观察，激发光波长488nm，功率15Mw，发射光波长560nm～615nm，用Laser Scanning Microscope 510 Version 1.5 软件对图象进行分析. 同时观察柔红霉素荧光在肿瘤细胞内的分布.

2 结果

2.1 柔红霉素在胃癌细胞内的分布 多药耐药细胞和亲代细胞各5×10⁵·孔^-1，经柔红霉素1.0mg·L^-1孵育1h后，激光共聚焦显微镜观察结果显示柔红霉素荧光主要集中在细胞核内，细胞质中亦有少量分布，且亲代细胞内柔红霉素的荧光强于耐药诱导细胞（图1）.

2.2 胃癌细胞对化疗药物的摄取 多药耐药诱导细胞和亲代细胞均为1×10⁶·孔^-1，［³H］VCR 37MBq·L^-1，1mL·孔^-1，孵育于37℃，每15，30，60，90，120min取2孔作同位素液闪测定. 结果表明，药物敏感和耐药细胞在细胞内［³H］VCR的浓度均随孵育时间的延长而增加，90min左右达到高峰，但耐药诱导细胞较亲代细胞对［³H］VCR的摄取缓慢，且达到高峰的浓度较后者低（图2）.

2.3 胃癌细胞对化疗药物的外排 经激光共聚焦显微镜观测结果显示，多药耐药细胞在撤去柔红霉素后15，30，45，60min时，细胞内的柔红霉素荧光强度均低于亲代细胞（表1）.

表1 胃癌细胞内柔红霉素的荧光强度（x±s）

细胞系	15min	30min	45min	60min
MKN45	127±16	115±9	65±13	36±9
MKN45/VCR1.0	122±17	65±10	42±3	34±4
SGC7901	142±30	117±39	85±20	69±32
SGC7901/VCR0.5	91±26	57±19	55±4	47±13
MKN28	176±26	129±25	104±24	78±13
MKN28/VCR1.0	127±18	84±9	59±7	45±8

3 讨论

在有关抗癌药物耐药性的文献中，报道典型的多药耐药（MDR）涉及的药物包括蒽环类、长春碱类、表鬼臼毒素类、秋水仙素等疏水性天然性产物，而烷化剂和抗代谢药物则不产生MDR^［^29-31^］. 多药耐药是由P-糖蛋白、多药耐药相关蛋白、肺耐药相关蛋白所介导的^［^32-41^］. 这些蛋白作为ATP依赖的药物泵，能主动地将药物从细胞内泵出，使得细胞对化疗药物的摄取减少，外排增加，从而导致肿瘤细胞内药物的浓度减少，细胞毒作用减弱^［^42-46^］. 我们对3种不同分化程度的胃癌细胞用长春新碱诱导产生的多药耐药细胞，利用［³H］长春新碱，采用同位素液闪测定结果证实3种耐药细胞均存在对化疗药物摄取的障碍.
利用柔红霉素在细胞内的自发荧光，应用激光全聚焦显微镜观测结果显示抗肿瘤药物主要集中分布在肿瘤细胞的核内，细胞质中有少量的分布，耐药细胞内药物浓度低于亲代细胞，撤药后动态观测显示3种耐药细胞对化疗药物的外排增加. 因而，此三种不同分化程度的胃癌细胞经长春新碱长期诱导后，均存在着对细胞毒药物摄取的减少，外排的增加，细胞内药物积累的障碍，从而导致对多种化疗药物的耐药. 这与Marquardt et al^［²³^］报道的HL60阿霉素耐药株和Breuninger et al^［²⁴^］报道的NIT 3T3耐药株存在的细胞对抗肿瘤药物积聚障碍等的报道嘁恢

图1 激光共聚焦显微镜观察柔红霉素在细胞内分布. 敏感细胞（A，C和E）和耐药细胞（B，D和F）孵育于含柔红霉素1.0mg·L^-1的培养液1h，MKN45细胞（A），MKN45/VCR1.0细胞（B），SGC7901细胞（C），SGC7901/VCR0.5细胞（D），MKN28细胞（E）和MKN28/VCR1.0细胞（F）内柔红霉素荧光分布显示该药物主要集中分布于细胞核内.

图2 胃癌细胞内［³H］VCR浓度变化曲线. 2a:MKN45和MKN45/VCR1.0细胞内［³H］VCR的cpm值随时间的延长逐渐增高，但耐药诱导细胞MKN45/VCR1.0内［³H］VCR的cpm值在各个时间点均低于亲代细胞；2b：SGC7901和SGC7901/VCR0.5细胞内［³H］VCR的cpm值随时间的延长逐渐增高，并于90min左右达到高峰，但耐药诱导细胞SGC7901/VCR0.5内［³H］VCR的cpm值在各个时间点均低于亲代细胞，且高峰时其值亦低于亲代细胞；2c：MKN28和MKN28/VCR1.0细胞内［³H］VCR的cpm值随时间的延长逐渐增高，并于90min左右达到高峰，但耐药诱导细胞MKN28/VCR1.0内［³H］VCR的cpm值在各个时间点和高峰时均低于亲代细胞.

经典的研究MDR功能的方法有2种^［^47-49^］：①通过同位素标记抗癌药物研究其在耐药细胞内的积聚；②利用rhodamine 123的荧光，用流式细胞仪检测耐药细胞内rhodamine 123的积聚间接地反映耐药细胞内抗癌药物的积聚. 激光全聚焦显微镜是通过对荧光物质的观察，运用计算机软件分析该荧光物质的荧光强度的方法^［⁵⁰^］. 我们利用柔红霉素在肿瘤细胞内的自发荧光，采用激光全聚焦显微镜观测的方法，避免了同位素标记价格昂贵及造成污染的缺点，又可以对药物分布进行定位和动态观测，同时可对观测到的荧光强度进行定量分析，弥补了普通荧光显微镜和荧光分光光度计的不足，成为细胞内药物代谢研究的一种有效方法.

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