【作者】 李金忠；

【导师】 李宁毅； 陈万涛；

【作者基本信息】 青岛大学 ， 口腔临床医学， 2004， 硕士

【摘要】 目的：拓扑异构酶Ⅱ在生物体内普遍存在，对细胞的DNA复制、转录，染色体的凝集和分离具有重要的作用，在增殖期细胞尤其是肿瘤细胞中表达增高，因而拓扑异构酶Ⅱ成为抗肿瘤药物的理想靶点。体外化疗药物敏感试验显示拓扑异构酶(Topo)抑制剂替尼泊甙(VM-26)对口腔恶性肿瘤的生长抑制作用显著优于目前常用化疗药物如顺铂、平阳霉素等。本研究目的是探讨替尼泊甙体内、外抑制口腔癌细胞增殖作用效果和作用机制，为该药物临床应用提供实验依据，并为寻求同类新药提供技术资料。 材料与方法：以人舌鳞癌Tca8113细胞系和人腭部小涎腺腺样囊性癌肺高转移ACC-M细胞株为研究对象。使用MTT检测法定量检测不同浓度VM-26及顺铂(Cisplatin，CDDP)对Tca8113细胞和ACC-M细胞的增殖抑制率：首先将Tca8113细胞和ACC-M细胞按一定密度接种于96孔板，在培养液中分别加入0.1μg／ml、0.5μg／ml、1.5μg／ml、5.0μg／ml、15μg／ml、45μg／ml的VM-26和0.1μg／ml、0.3μg／ml、1μg／ml、3μg／ml、9μg／ml的CDDP，继续培养1 d、2 d、3 d、4 d、5 d，然后加入MTT，形成结晶后，以二甲基亚砜(DMSO)充分溶解结晶，酶联酶标检测仪测定其吸光度(Optical Density，OD)。nu／nu BALB／C裸鼠36只，鼠龄5周，SPF级条件饲养，称重后分层随机分为两大组，每组18只。一组每只右侧腋下部的皮下接种Tca8113细胞移植瘤组织块约8mm~3大小；另一组每只手左侧腋下部皮下接种ACC-M细胞移植瘤组织块8mm~3大小。7天后，两组动物分别随机分为三个亚组：对照组、VM-26处理组和CDDP处理组；每组6只。每亚组动物分别腹腔注射生理盐水、VM-26和CDDP，VM-26剂量为10mg／kg／次，CDDP中文摘要剂量为sm叭留次，每3天用药1次，共3次。每3天记录肿瘤大小和动物的一般情况。用药35天后引颈处死动物，称量动物体重和瘤重。 选择Tcas 113细胞为实验对象，进行vM一26作用机理研究。先使用荧光染色法观察VM一26作用下细胞生存情况，在Tcas 113细胞培养液中分别加入0.1林g/ml、0.5林g/ml、1 .5林g/ml、5.0林g加1、15林g/ml、45林g/ml的VM一26药物，继续培养ld、Zd、3d、 4d、sd，然后加入EB/AO荧光染料染色细胞，荧光显微镜观察细胞生存情况，并照相;收集同法处理的Tcas 113细胞、以2%戊二醛磷酸盐固定液等固定、脱水包埋、超薄切片、醛酸铀和柠檬酸铅双染色，透射电镜观察其超微结构下的形态变化，照相记录。收集0.15林g/ml，1.5林g/ml，5.0卜g/ml和15林g/ml浓度vM一26作用48h后的Tcas 1 13细胞，PBS漂洗，预冷的70%PBS乙醇固定，PBS洗，RNase处理后以PI染料避光染色30min;200目尼龙网过滤后，流式细胞仪检测其细胞周期分布。以AlmexinV试剂处理上述VM一26干预的几as 1 13细胞，应用FeM检测细胞的凋亡率。选择0.15林g/ml和5.0林g/ml两个浓度的VM一26作用12h、24h、36h、48h和72h的Tcasll3细胞，同上述方法使用流式细胞仪检测其细胞周期分布和凋亡率。实验重复3次，取平均值，均数间比较采用t检验，p<0.05时有显著性差异。结果:MTT法检测结果表明，VM一26、CDDP对Tcas 113细胞和ACC一M细胞的生长抑制率呈明显的量效关系和时效关系。VM一26(5，o林留ml)和cDDP( 3.0林留ml)作用Tcas 113细胞72h，生长抑制率分别达到92.19%和58.9%。VM一26和CDDP对Tcas 113细胞的半数致死剂量分别为0.35林留ml和1 .1林g/ml。荷Tca8113细胞动物的对照组、vM一26处理组和顺铂处理组的平均肿瘤体积分别为3146.50mm3、3巧.08m砰和1225.43~3;荷ACC一M细胞动物的肿瘤平均体积分别为，对照组473 1 .1 7mm3、vM一26组1 1 84.13mm3和CooP组2688.35mm3。vM一26和CooP对Tcas 113移植瘤生长抑制率分别为75 .24%和55.59%，两药物对ACC一M移植瘤的抑制率分别为72.45%、39.65%。两处理组动物未见严重的不良反应发生。荧光染色可直观地表现出Tcas 113细胞随VM一26浓度的上升和作用时间的延长诱导凋亡程度加强。TEM观察可见大部分细胞核浆比例变小、核质浓缩，核膜皱缩，同时染色质固缩，呈块状或新月形位于核膜下，细胞膜结构完整，呈现典型的细胞凋亡形态。流式细胞仪检测结果表明，5.0林g/ml和15林创ml浓度的VM一26中文摘要处理Tcas 113细胞可产生明显的细胞凋亡，48h后细胞凋亡率均分别可达77.5%和81.2%，细胞由Gl/GO期逐渐聚集于S期，表现为GI/GO期峰逐渐向二倍体和四倍体之间移动并停止在二者中间，同时峰高度变低，基底逐渐增宽，显示此时细胞内DNA含量不均一;0.15林岁ml浓度的VM一26处理12h，Tcas 113细胞的周期分布即可发生变化，表现为细胞逐渐通过S期，最终聚集在GZ/M期而不再发生变化，该期细胞比例从对照组细胞的10.75%上升到药物作用72h时的75.18%，但其凋亡率同5.0林g/ml vM一26组的Tca8113细胞明显为低，即使作用72h，凋亡细胞比率也不超过30%。结论:1、体外条件下VM一26对口腔肿瘤细胞较CDDP具有更高的生长抑制作用。2、动物体内实验显示，VM一26对口腔肿瘤细胞也可产生显著的肿瘤抑制作用。VM一26应用具有较好的安全性，提示VM一26是治疗口腔癌(鳞癌、腺癌)安全、有效的药物。3?更多还原

【Abstract】 Objects: As is well known that topoisomerase II exist in cells ubiquitously and play an essential role in DNA replication, transcription, chromosome formation and separation of sister chromosome. This enzyme express in a higher level in proliferating cells, especially in malignant tumor cells. So, it has been an ideal target for antitumor drugs. Both clinical findings from clinical chemotherapy to patients with oral and maxillofacial malignant tumor and in vitro chemosensitivity testing with MTT method showed that the antitumor effect of teniposide (VM-26), a kind of topoisomerase II poison, are more significant than those of other chemotherapeutic drugs like cisplatin (CDDP). Present study is designed to identify quantitatively the effect of VM-26 both in vitro and in vivo and furthermore, to explore the underlying mechanism, and the findings will be used to indicate the usage of this drug in clinical practice.Material and Methods: Human tongue squamous cell carcinoma cell line (Tca8113 cell) and cell strain with high metastatic ability to lung induced from human adenoid cystic carcinoma (ACC-M cell) were used as subjects in this study. We firstly tested quantitatively the inhibition rate of tumor cell proliferation by VM-26 using MTT testing, with CDDP as control drug: Tca8113 cells and ACC-M cells were inoculated in 96 wells micro culture plate at a concentration of 5 104/well and 4 104/well and cultured for 24 h.Then, VM-26 of 0.1 g/ml, 0.5 g/mK 1.5 g/ml, 5.0 g/ml, 15 g/ml, 45 g/ml and CDDP of 0.1ng/ml, 0.3 g/ml, 1 g/ml, 3 g/mk 9 g/ml were added in culture medium. Cells were continued to be cultured for 1 d, 2 d, 3 d, 4 d, 5 d. Supernatants were aspirated and MTT was added in each well after culture for certain time. DMSO was added in to dissolve crystals and the ODs of these solutions were tested using UV spectrometer. 36 nu/nuBALB/C nude mice, aged 5 weeks, fed in thespecific pathogen free (SPF) condition, were divided randomly into 2 groups, 18 mice in each group. Mice in one group were inoculated with xenografts of Tca8113 cells of 8 mm3 in size, in the right armpit region and mice in the other group were inoculated with xenografts of ACC-M cells, 8 mm3 in size, in the left armpit region. 7 days later, each group was divided into 3 subgroups: control group, group treated with VM-26 and group treated with CDDP, 6 mice in each group. Mice in each subgroup were injected intraperitoneally with normal saline, VM-26 of 10mg/kg and CDDP of 5mg/kg respectively, once every 3 days and total 3 times. The volumes of tumors and general conditions of mice were recorded every 3 days. Tca8113 cells were selected as subject in further study on mechanism of VM-26 killing tumor cells. EB/AO fluorescent staining reagent were added in cells culture medium having been treated with VM-26 of 0.1 g/ml, 0.5 g/ml, 1.5 g/ml, 5.0 g/ml, 15 g/ml and 45 g/ml for 1 d, 2 d, 3 d, 4 d and 5 d, and cells were observed using fluorescent microscope. On the other hand, such cells were collected, fixed with 2% glutaraldehyde in PBS, dehydrated, embeded, chiped, double stained with lead citrate and uranyl acetate, observed its apoptosis using TEM and recorded in photographs. Tca8113 cells having been treated with VM-26 of 0.15 g/ml, 1.5 g/ml, 5.0 g/ml and 15 g/ml for 24 h, were collected and washed with PBS, fixed with cold 70% ethanol in PBS, washed in PBS again, treated with RNase to eliminate RNA and stained with PI in dark. Then, cells were filtered through 200-pore nylon membrane and collected using FCM. Cells distribution in cell cycle was analysed using Mod Fit software. Cells treated as above were stained using reagents in Annexin V kit, and acquired using FCM to test their apoptosis rate. Cells having been treated with VM-26 of 0.15 g/ml and 5.0 g/ml for 12 h, 24 h, 36 h, 48 h, and 72 h, were tested cell cycle distribution and apoptosis with FCM as above. This experiment was repeated in triplicate and the average values were acquired.Results: Findings from MTT assay showed VM-26 and CDDP inhibit the proliferations of tumor cells both i更多还原