【作者】 代智；

【导师】 仲来福；

【作者基本信息】 大连医科大学 ， 劳动卫生与环境卫生学， 2003， 硕士

【摘要】 前言 顺铂（cis-diamminedichloroplatinum，CDDP）是当前临床使用的主要抗癌药物之一，治疗卵巢癌、睾丸癌、膀胱癌及头颈部癌等有很好的疗效。但是，CDDP作为一种细胞毒性药物，在治疗肿瘤的同时，亦可对正常组织如肾脏、周围神经等造成损害，其中，肾毒性是限制其临床用药剂量的主要因素。有关CDDP细胞毒性的机理尚不十分清楚。一种观点认为，脂质过氧化损伤是CDDP细胞毒性的机理之一，CDDP可通过产生活性氧自由基（reactive oxygen species，ROS），耗竭细胞内还原型谷胱甘肽（GSH），抑制抗氧化物酶活性，最终造成肾毒性；另一种观点认为，CDDP可与细胞内DNA结合生成Pt-DNA加合物，这种加合物可抑制DNA合成，促使细胞凋亡发生。本研究从细胞、分子水平应用生物化学、分子生物学技术探讨CDDP细胞毒性机制，为进一步阐明CDDP毒性机理提供实验依据。大黄素（emodin，EMD），是中药大黄和何首乌中的有效成分，具有抗病毒、抗炎、抗肿瘤、抑菌、扩张血管、免疫抑制等多种药理作用。研究表明，EMD能增强正常细胞经CDDP诱导的DNA损伤的修复，起到预防作用，其中，切除修复基因ERCC1表达增强和Ca²⁺调节可能参与这一修复过程。EMD对CDDP细胞毒性预防作用其他机制方面的研究很少。本研究从CDDP损伤机制入手探讨EMD的预防作用机理，这为今后更深入研制能减少CDDP毒副作用新药做了很好铺垫，同时也为临床工作者更好的应用EMD提供了科学依据。 方法 本实验以体外培养的人胚肺成纤维细胞WI-38作为研究对象。将WI-38细胞以一定浓度接种至培养瓶或培养板内，待细胞生长至融合状态时，给予不同浓度的CDDP处理，然后于37℃、5％CO₂孵育箱内培养一段时间后，通过光镜、电镜观察CDDP引起的细胞形态学及超微结构改变，噻唑蓝（MTT）法检测CDDP细胞毒性，荧光法测定氧自由基水平、细胞内脂质过氧化物水平、GSH含量以研究CDDP引起的氧化性损伤，通过琼脂糖凝胶电泳及流式细胞仪技术研究CDDP引起的DNA损伤。同时，又以EMD为干预物用上述方法测定其对CDDP引起细胞氧化性损伤和DNA损伤的预防作用，研究其预防机制。结果倒置显微镜下观察，发现wi一38细胞经60、120mg·L一，CDDP处理巧h后，细胞逐渐圆化，胞膜边界清晰，并随CDDP浓度的增加改变愈加明显;30 mg屯一，EMD干预后，细胞质逐渐伸展。经MTT法测定，CDDP作用22h后对W工一38细胞毒性最大，半数抑制浓度(IC动为16士3 mg·L一，;30mg·L一，EMD干预后，细胞毒性明显减小，ICS。为34士6mg·L一，。经荧光法测定，CDDP作用不同时间后，细胞内氧自由基水平明显增加，且在作用3h后，增加最明显，此时GSH含量明显减少，CDDP作用22h后，细胞内脂质过氧化物水平明显增加;EMD干预后，细胞内氧自由基、脂质过氧化物增加更加明显，GSH减少也更加明显。透射电镜下观察发现，CDDP作用22h后，部分细胞表面形成较大的胞质突起，胞质内含有大大小小的空泡，核内异染色质边集于核膜;EMD干预后，此现象基本消失。经琼脂糖凝胶电泳测定，CDDP作用22h后，呈现典型180²00 bp整数倍的梯形条带，并随浓度增加条带愈加明显;EMD干预后，梯形条带几乎消失。经流式细胞仪检测，发现CDDP使细胞凋亡率明显增加，G。十G:期细胞百分数明显减少，S期细胞百分数明显增加;EMD干预后，细胞凋亡率明显减少，G。十G。期细胞百分数增加，S期细胞百分数减少。结论（1）CDDP可诱导细胞产生氧自由基，耗竭细胞内GSH，脂质过氧化物生成增加，诱发氧化应激，引起细胞氧化性损伤。（2）CDDP可导致细胞内DNA损伤，使细胞周期改变，从而诱发细胞凋亡。（3） EMD不仅不能减少CDDP对细胞的氧化性损伤作用，而且能协同CDDP增强其对细胞的氧化性损伤。（4） EMD对CDDP细胞毒性的预防作用可能与减少DNA损伤的发生和对细胞周期调节的影响有关。更多还原

【Abstract】 Introduction Cisplatin (cis-diamminedichloroplatinum, CDDP) is one of the primary anticancer drugs widely used in the clinic. It has apparent clinical effects on testicular cancer, ovarian cancer and so on. However, CDDP, as a kind of cytotoxicity drug, also can produce some side effects on kidney, peripheral nerve and other normal tissues, which hinders its clinical uses. Untill now, the mechanisms of CDDP-induced cytotoxicity have been unknown. It has been reported that oxidative stress and lipid peroxidation is one of the mechanisms of CDDP-induced cytotoxicity. CDDP can cause kidney damages by producing reactive oxygen species (ROS), exhausting cellular glutathione (GSH) and inhibiting the activity of antioxidant enzyme; Also, CDDP can bind to DNA base and form a stable Pt-DNA complex which will disturb the structure of DNA, inhibit DNA replication and thus lead to apoptotic evidence. In this study, we will explore the mechanisms of CDDP-induced cytotoxicity by using biochemistry and molecular biology technique. Emodin (EMD), usually extracted from rhubarb, is an active component. It has biologically extensive effects. It has been reported that EMD can elevate ERCC1 gene expression and thus increase the repair of cisplatin-induced DNA damages in human cells. In addition, Ca2+-mediated DNA repair process may be involved in the repair mechanisms of EMD. In this study, we will study mechanisms of EMD’s preventive effects on cisplatin-induced cytotoxicity and provide scientific evidence of making wide use of EMD in the future. Methods In this study, human lung diploid fibroblast cells (WI-38) were used as experimental objects. WI-38 cells were seeded in culture plate or flask and incubated at 37 癈 for at the least 24 h prior to use. Then serial amounts of CDDP were added to the wells and flasks. WI-38 cells were incubated continuously at 37℃ for some time. With the help of optical and electron microscopy, morphology and ultrastructurechanges of WI-38 cells were observed. Cytotoxicity was determined by MTT color assay. ROS, lipid peroxidants and GSH were measured by fluorescence method. DNA damages were determined by electrophoresis and flow cytometry. With the help of the methods described above, HMD as an intervenor was added into cultures together with CDDP to explore its preventive effects on oxidative damages and DNA damages induced by cisplatin.Results After WI-38 cells were incubated with cisplatin for 15 h, morphologicalchanges were apparently observed and the changes were alleviated by HMD. AfterWI-38 cells were treated by different concentrations of CDDP for 22 h, CDDPproduced the strongest cytotoxicity and IC5o was 16±3 mg L-1; The cytotoxicityinduced by CDDP was significantly decreased by HMD and IC50 was 34 ± 6 mg L-1. Theproduction of reactive oxygen radicals was significantly increased after WI-38 cellswere treated by CDDP for a period of time and the increase was the most significantafter CDDP treatment for 3 h. At the same time, GSH level was significantly decreased.Lipid peroxidants were also significantly increased after WI-38 cells were treated byCDDP for 22 h. The production of lipid peroxidants and ROS was further increased byHMD and GSH level was decreased. With the help of electron microscopy, we foundthat cellular ultrastructure has been changed. After WI-38 cells were treated by CDDPfor 22 h, larger cytoplasm protuberances turned up on some part of the cell surface,many vacuoles appeared in the cytoplasm and chromatin was peripherally aggregated.The change could be remarkably inhibited by HMD. DNA electrophoresis showed thatthe ladder bands, a typical feature of apoptotic cell, appeared in the groups treated byCDDP for 22 h, and with the increase of dosage the phenomena was more apparent;the DNA damage could be alleviated by HMD. Flow cytometry showed that theapoptotic rate was significantly increased, at the same time, the cell numbers in Sphase were increased and the cell numbers in G0±G1 phase were更多还原