【作者】 阴梅云；

【导师】 阎蕴力；

【作者基本信息】 河北医科大学 ， 人体解剖与组织胚胎学， 2003， 博士

【摘要】 目的 卵巢癌化疗中，顺铂(cisplatin, CDDP)应用普遍，但相关抗药性问题也越来越突出。因此，选择能有效与之配伍的药物是临床探索的问题之一。阿克拉霉素(aclarubicin, ACR)作为拓扑异构酶Ⅱ(topoisomeraseⅡ,topoⅡ)抑制剂之一，在卵巢癌的治疗中使用较少，并且与CDDP合用时相互作用机理尚不明确。为此，本研究通过ACR与CDDP联合用药的体外实验，旨在探讨卵巢癌化疗药物组合，为筛选有效抗肿瘤药物提供依据。材料和方法1材料 卵巢浆液性囊腺癌（SKOV3）细胞株引自北京大学人民医院。ACR为深圳万乐药业有限公司产品。CDDP为昆明三戎金属药业有限公司产品。免疫细胞化学试剂，一抗为鼠抗人topoⅡ抗体（美国Sandcruz公司），辣根过氧化物酶(HRP)标记的生物素化二抗购自武汉博海生物公司。RPMI-1640培养基、噻唑蓝(MTT)、RNA酶、蛋白酶K、中分子量蛋白标准、吖啶橙、嗅乙啶购自华美公司。2 方法2.1 细胞培养和药物作用浓度选择SKOV3细胞以RPMI-1640培养基培养，内含10%新生牛血清、青霉素100 U/ml、链霉素100μg/ml，置37℃、5% CO2环境下培养。应用药物的血浆峰浓度(peak serum concentration, PSC)，其中ACR为6μg/ml；CDDP为3μg/ml进行相应实验分组。2.2 MTT方法检测药物对细胞的抑制率取指数生长期细胞，以1×105/ml细胞浓度接种于96孔培养板（100 μl/孔），加入无血清培养液稀释的药物10μl。实验组将ACR的PSC浓度(6μg/ml)分别与CDDP的1/10 PSC(0.3μg/ml)、PSC浓度(3μg/ml)、10×PSC浓度(30μg/ml)合用。同时设对照样品。不同药物及浓度均为3复孔。药物与细胞共同培养至24h， 每孔加<WP=5>入5mg/ml MTT 20μl。继续37°C孵育4小时后，离心吸去上清，加入200μl /孔DMSO终止反应。用酶标仪测570nm波长吸光值(A值)。计算抑制率，按金氏公式分析ACR与CDDP合用对卵巢癌细胞抑制效应。本实验重复2次。2.3 双荧光染色观察凋亡细胞将细胞与药物共同作用24h后，经1500rpm/min离心5分钟，悬浮于磷酸缓冲液中。配制吖啶橙和嗅乙啶各为0.5mg/ml 的混合染液。加荧光染液于细胞悬液内，吹打混匀、滴至载玻片上，置荧光显微镜下观察，每个样品定量计数300个细胞。按公式：凋亡细胞数 / 总细胞数×100%，计算调亡率，用按金氏公式分析实验结果。2.4 克隆抑制率检测 将细胞按1×105/ml浓度接种于培养瓶。细胞进入指数生长期后加入药物，每组均为3个实验瓶。药物与细胞共同孵育24h后，消化细胞，无血清培养液清洗。按1×103/ml细胞加入60mm2培养皿培养。连续培养7天后，0.5%结晶紫染色，镜下计数各组克隆形成数。按公式：（1-克隆形成数 / 接种细胞数）×100%，计算克隆抑制率，按金氏公式分析结果。2.5 DNA提取及电泳药物与细胞共孵育24h。离心收集细胞，加入细胞裂解液，4℃裂解20 min。经RNA酶(50μg/ml)37℃作用1h，蛋白酶K 50℃作用3h。以酚-氯仿法抽提DNA，70%乙醇洗涤后凉干。DNA沉淀溶于20μl TE溶液(10 mmol/L Tris-HCl, 1mmol/L EDTA)，内含1mg/ml嗅乙啶。行1.5%琼脂糖凝胶电泳。紫外透射仪观察、记录。2.6 蛋白印迹分析topoⅡ表达收集各组细胞经磷酸盐缓冲液洗涤后，加入200μl预冷细胞裂解液4℃裂解40 min。经蛋白定量后，将10μg样品加入等量上样缓冲液，经10% SDS-PAGE电泳后，用半干式电转移仪转至硝酸纤维素膜上。加入一抗，鼠抗人topoⅡ抗体4℃孵育过夜。羊抗鼠HRP标记二抗，37℃孵育1 h。二氨基联苯胺(DAB)显色后分析结果。2.7 免疫细胞化学检测泛素表达<WP=6> 药物作用后离心收集细胞、经4%多聚甲醛固定后，滴至涂有多聚左旋赖氨酸的载片上。用冷0.1M PBS (pH 7.4)冲洗。3%甲醇-过氧化氢室温孵育15 min封闭过氧化物酶。1%Triton X-100，40C过夜。冷PBS冲洗3遍，5min/次。湿盒内10%山羊血清孵育30 min，370C。滴加1:200(0.01M PBS稀释)一抗，小鼠抗人泛素单克隆抗体，于湿盒内40C孵育过夜。其后PBS清洗三次，5min/次；滴加二抗，羊抗小鼠生物素IgG(1%BSA-PBS稀释)，湿盒内370C温育30 min。PBS冲洗后，滴加1:300稀释(0.01M PBS稀释) HRP标记的亲和素，370C温育40min。滴加DAB显色。苏木精复染，常规脱水、透明、封片、显微镜下观察记录。结 果1 MTT检测结果应用MTT检测可见：ACR单独作用后对卵巢癌抑制率为12.5±1.2%，CDDP单独用药依据作用浓度的不同，0.3μg/ml浓度下抑制率为6.20±3.2%；3μg/ml为23.5±1.2%，30μg/ml为56.3±2.2%，而与ACR共同作用后，其抑制率分别提高为18.5±4.3%、35.7±5.1%、78.2±4.0。以上结果用金氏公式分析两药的合并效应，结果表明，ACR与CDDP具有明显的协同效应。2 双荧光染色观察细胞凋亡将药物与细胞共同作用后，经吖啶橙、嗅乙啶双荧光染色观察可见：正常细胞呈绿色，依据常染色质和异染色质不同，荧光密度有所差异。凋亡细胞或可见凋亡小体，中晚期凋亡细胞呈橘红色或红色、染色质凝集、染色均匀，细胞核变小。计算各组细胞凋亡率，结果表明：ACR和CDDP合用与药物单用相比，细胞凋亡率明显增加。3 药物对细胞克隆抑制率的影响为判断不同药物作用后细胞的生存能更多还原

【Abstract】 Objective Cisplatin (CDDP) is frequently used anticancer agent in the treatment of ovarian cancer, but the problem on the drug-resistance to CDDP is becoming more and more obviously in clinic. So it is urgent to find some drugs that can combine with CDDP to therapy the cancer more effectively. Aclarubicin(ACR), one of the topoisomeraseⅡ(topoⅡ) inhibitors, is less used in the ovarian cancer chemotherapy, also it is not cleared about the interaction between ACR and CDDP. This in vitro study on the mechanisms of ACR combined with CDDP to inhibit ovarian cancer cell was carried out on attempt to form a promising class of agents, and hope that might be helpful for improving treatment efficiency over the cancer.Materials and Methods1 Material The SKOV3 ovarian cell line was supplied by the Peking people’s hospital. ACR is product of the WanLe Drug Co. Ltd. Shenzhen, China and CDDP is produced by Sanrong Co. Ltd. Kunming, China. The immunocytochemical reagents, a mouse anti-human topoⅡprimary antibody was purchased from Sandcruz Co. American, and a goat anti-mouse biotin-horseradish peroxidase (HRP) secondary antibody was bought from Wuhan Boster Biological Technology Co., Ltd. RPMI-1640 culture medium (Gibco-BRL), microculture tetrazolium (MTT), RNase, Proteinase K, protein marker were purchased from Sino-American Biotechnology Co.2 Methods2.1 Cell culture and the concentration selection of the drugs<WP=12> The SKOV3 cells were cultured in a humidified atmosphere of 5% CO2 at 37 °C in RPMI 1640 medium containing 100U/ml penicillin, 100μg/ml streptomycin and 10% newborn calf serum(NCS)The peak serum concentrations(PSC) of ACR(6μg/ml) and CDDP(3μg/ml) were chosen to form the experimental groups.2.2 MTT Assay Exponentially growing SKOV3 cells were added at 1x 105/ml in 96-well microtiter plates (100 μl/well). The drugs diluted by RPMI 1640 culture medium without NCS were added to the cells in a final volume of 10 μl/well. The PSC of ACR(6μg/ml) was combined with 1/10 PSC(0.3μg/ml), the PSC(3μg/ml) and 10×PSC(30μg/ml) of CDDP, respectively. Three replicates were made for each concentration of the drugs. After drugs exposure for 24h, 20 μl of MTT compound was added to each well and the cells were incubated at 37°C for 4 h. Following the plates were centrifuged and discarded supernatant, the cells were then incubated with 200 μl of dimethyl sulfoxide to stop the reaction. Absorbance (A value) was read at 570 nm using a microplate reader. The inhibition ratios were measured and the synergistic effectiveness of the two drugs was calculated by using the jin’s formula. Two separated experiments were made for MTT test. 2.3 Determination of cellular apoptosis by double fluorescent dyes After incubation for 24h, the cells were centrifuged by 1500rpm/min for 5min and suspended in phosphate-buffered saline (PBS). The cells were then mixed with a fluorescent dye mixture (acridine orange 0.5mg/ml and ethidium bromide 0.5mg/ml) and added to slides, which were detected under a fluorescence microscope. To assess the percentage of cells showing features of apoptosis, at least 200 cells were scored in each experiment. The apoptotic ratios were measured by using the formula, numbers of apoptotic cells/ whole cells×100. Statistical significances were determined by the jin’s formula.2.4 Colony inhibition assay <WP=13> Briefly, 1×105 /ml cells were seeded on cell culture flasks. Three flasks were prepared for each group. Following the drugs were added to the exponentially growing cells and exposed for 24h, the cells were centrifuged and washed with the culture medium without NCS, then replanted at a density of 1×103 to 60mm2 dishes. The colonies were counted 7d later after staining with 0.5% crystal violet/ethanol. The colony inhibition efficiency (%) were calculated by using a formula of (1-apoptotic cells / planted cells)×100. The differences between the results were tested for statistical significance by the jin’s formula. 2.5 DNA Isolation and Electrophoresis更多还原