【作者】 陈新志；

【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】 在化学物对肾脏造成的损伤中，DNA损伤被认为是其机制之一。顺铂（顺式二氯二氨铂，Cis-diamminedichloroplatinum，CDDP）是临床最主要的化疗药物之一，但往往因其严重的肾脏毒性限制了其用药剂量。CDDP肾毒性的机制之一是氧化应激，但是氧化性损伤并不能完全解释顺铂诱导的肾毒性。人们在研究中发现顺铂肾毒性另一个重要机理是DNA损伤。顺铂可与DNA能形成链内、链间交联，顺铂肾毒性则与铂（Pt）同DNA形成加合物的水平有关。镉肾脏毒性也与氧化损伤有关，可能是引起细胞毒性的一个重要机制。但也有实验证据表明氧化性损伤并非镉引起肾损伤的直接原因。镉可引起DNA链断裂，形成DNA碱基修饰产物8-羟脱氧鸟苷，损害DNA修复系统，从而造成DNA损伤。马兜铃酸所致肾损伤机制涉及DNA损害、肾小管细胞转分化等。马兜铃酸与DNA形成加合物可能是其肾毒性和致癌性的重要机制。 在DNA损伤的众多修复途径中，核苷酸切除修复（NER）途径能广谱识别大多数严重损伤造成的DNA螺旋扭曲，是DNA损伤的主要修复系统，也是机体抵抗致癌效应的主要防御机制。对大分子加合物来说，只有NER途径具有修复活性。NER的过程涉及特异性损伤的识别，损伤链的双酶切，损伤部位的移去，缺口的填补和最终链的连接。 本研究运用了分子生物学手段利用两步转染法将携带NER基因ERCC₁/XPF的重组质粒转染至人胚肾293细胞，建立了过表达ERCC₁/XPF蛋白的细胞系，以提高293细胞的NER能力。并以此为研究对象观察了上述三种肾毒物对293细胞和过表达ERCC₁/XPF蛋白293细胞的细胞毒作用，以阐明三种肾毒物致活细胞DNA损伤在其各自细胞毒性机制中所处的地位，并评价这种损伤的程度。用NER系统来评价肾脏毒物致活细胞DNA损伤的程度以及阐述肾毒性机制，作者尚未见国内外报道。 方法 本课题利用两步转染法将携带核苷酸切除修复基因ERCC₁和XPF的重组质粒转染至真核细胞人胚肾293细胞。然后用Zeocin和G₄₁₈筛选获得细胞克隆，扩增后再通过W七stem七lot检测目的蛋白的表达。采用顺铂对转染前后细胞形态学和细胞毒作用的影响来评价表达蛋白的生物学功能。 MTT比色法检测顺铂、氯化锡和马兜铃酸染毒1小时对转染前后细胞在24小时的抑制率，Bliss法计算半数抑制浓度（I C50）。 结果W七stern一blot检测出有目的蛋白的过表达。在相同浓度顺铂作用下，转染后细胞的受损情况于光镜、电镜和荧光显微镜下观察均有明显减轻。不同浓度顺铂对转染后细胞·的抑制率明显低于293细胞，表明成功获得了稳定转染ERCC;/Xl，F的ERCCI/X卫F一293细胞株。 受试物处理细胞1小时，温育24小时后用MTT法检测三种肾毒物对ERCCI/XPF一293和293细胞的ICS。，结果显示顺铂、氯化锡和马兜铃酸对ERCC:乃JF一293细胞的IC，。均高于293细胞。顺铂对ERCCI/XPF一293和293细胞的ICS。分ZfJ为1 .0 11和0.2叫mmo比（P<0.05）。氯化锡对ERCCI/xpF一293和293细胞的ICS。分别为61.91和15.61 p mo比（P<0.05）。马兜铃酸对ERCCI/X卫F一293和293细胞的ICS。分别为321.7和201.9 p mo比（P<0.05）。 结论经两步转染成功建立了过表达ERCC;/XPF蛋白的ERCCI/XPF一293细胞株，过表达的ERCC[次卫F蛋白具有生物学功能，能引起活细胞整体NER能力的升高。由于NER能力的升高能降低顺铂、氯化锅和马兜铃酸细胞毒细胞毒性，提示这些肾毒物都能导致活细胞DNA损伤并且所致这种DNA损伤是其肾毒性的重要机制。在本研究条件下三种肾毒物所致的DNA损伤对活细胞来说都是一种严重损伤，靠自身的修复系统不易修复，升高的NER系统则能不同程度的予以修复。更多还原

【Abstract】 DNA damage is thought of one mechanism of chemicals-induced nephrotoxicity Cisplatin (Cis-diamminedichloroplatinum, CDDP), one of primary anticancer drugs, hinders its clinical uses for its nephrotoxicity. It has been reported that oxidative stress was one of the mechanisms of CDDP-induced cytotoxicity. Besides these, CDDP can bind to DNA base as the target and form a stable Pt-DNA adducts which will disturb the structure of DNA and inhibit DNA replication. CDDP-induced nephrotoxicity has a linear correlation with Pt-DNA adducts level. Cadmium(Cd) causes oxidative damage, which is closely associated with the nephrotoxicity of Cd. But some studies provide evidences that oxidative damage is not a direct reason of Cd-induced nephrotoxicity. Cd can cause DNA damage by inducing DNA strand breaks, forming 8-hydroxydeoxyguanosine and inhibiting DNA repair. Aristolochic acid (AA) damages DNA and forms DNA adducts, which may be one of the important mechanisms causing its nephrotoxicity and carcinogenesis.Nucleotide excision repair (NER) can recognize a broad spectrum of helix-distorting lesions and is a major repair system of DNA damage. NER is the sole DNA repair activity for removing bulky adducts and a major defense against the carcinogenic effects. NER consists of removal of the damaged nucleotide(s) from DNA by dual incision of the damaged strand on both sides of the lesion, followed by filling of the resulting gap and ligation.To study whether CDDP, Cd and AA -induced DNA damages play the role in the mechanisms of their nephrotoxicities and to provide experimental evidences in live cell, an over-expressing ERCC1/XPF protein model cell strain was established.Methods The human ERCC1 and XPF gene were coexpressed in 293 cells by using biochemistry and molecular biology technique. The colony-like transfected cells were obtained after being selected with Zeocin and G418. The expression of ERCC1 and XPF was detected by western-blot after the clone cells were expanded. Toinvestigate the morphology changes and cytotoxicity after being exposed to CDDP to testify whether the cell strain which can over-express ERCCj/XPF protein was successfully established.The cytotoxicities of CDDP, cadmium chloride(CdCl2) and AA to different cells were measured by MTT assay. IC50 and its 95% confidence limit were calculated by using Bliss method and used to make statistical deducing.Results There were over-expression of ERCC1/XPF in the ERCC1/XPF-293 cells by Western-blot. The morphology changes and cytotoxicity after being exposed to the same concentration of CDDP were markedly alleviated. Those indicated that the cell strain which can over-express ERCCi/XPF protein had been established successfully.Inhibitory effects on transfected cells decreased after CDDP, CdCl2 and AA treatment, respectively. The IC50 of CDDP for 293 and ERCCi/XPF-293 cells were 0.294 and 1.011 mmol/L, respectively (P<0.05). The IC50 of CdCl2 for 293 and ERCC1/XPF-293 cells were 18.61 and 61.91 U mol/L, respectively (P <0.05). The IC50 of AA for 293 and ERCC1/XPF-293 cells were 201.9 and 321.7 umol/L, respectively (P <0.05).Conclusion The human ERCC1 and XPF gene can be coexpressed in 293 cells by using biochemistry and molecular biological technique, over expressing ERCC1/XPF would increase DNA repair capacity which can decrease the CDDP, CdCl2 and AA-induced cytotoxicities. It indicates that DNA damage is a critical role in the genesis of their nephrotoxicities.更多还原