【作者】 张军；

【导师】 于红刚；

【作者基本信息】 武汉大学 ， 内科学， 2013， 博士

【摘要】 背景顺铂是一个中性、无机平面正方形的复合物,其与DNA发生反应,诱导其产生特有的生物学效应,从而发挥修复的DNA损使细胞生存或激活不可逆的凋亡程序,导致细胞发生凋亡。然而,顺铂与DNA发生相互作用,主要通过一系列的自发水合反应,这其中涉及与水分子取代cis-chloro配体的过程[2]。这种单水化的形式被确认为高反应性的一种形式,但这种形式形成率往往受许多内源性亲核物质所限制,如：谷胱甘肽(GSH),甲硫氨酸,金属硫蛋白,和蛋白质等。因此,顺铂进入细胞时,它具有潜在的易受以上这些物质以及其他细胞成分的影响而导致细胞质灭活。细胞毒类化疗药物顺铂,其主要与亲核DNA中的嘌呤碱基N7位点发生反应,从而形成DNA-蛋白质和DNA-DNA链间和链内交联等复合物形式。然而,有证据表明形成的链内加合物可以诱导细胞毒作用的发生。另外,有报道说在培养的细胞中发现一个类似于链内交联DACH-硫酸根-铂的复合物,此复合物主要是针对不同数量和质量的DNA碱基的区别。从而为可能杀伤此类导致顺铂产生耐药性的肿瘤细胞细胞提供新的临床思路。由于形成的链内DNA加合物可以引起大量的顺铂诱发的核损伤,因此铂顺铂与DNA结合的水平和程度决定的细胞毒性。虽然顺铂影响DNA复制,但其影响DNA合成和细胞毒性的产生之间不存在相关性。因此,每一个识别蛋白可能会启动一个或多个特定的生物信号应激源,从而使DNA损伤呈现出看似无关的生物效应,此与加合物顺铂破坏的复制和转录过程是一致的,但这样的生物效应不一定直接导致细胞死亡。对顺铂作用的细胞来讲,其生存和促凋亡信号同时被激活,同时顺铂作用相对强度和/或持续时间来激活并整合下游的信号分子,最终产生不同的生物学效应,并导致顺铂耐药性的产生。结直肠癌是临床上比较常见的恶性肿瘤,其发病率居第三位,死亡率较高[8]。时至今日,治疗肿瘤的方法不断发展与更新,新的抗癌药物层出不穷,但顺铂仍是临床上较常用的化疗药物,尤其是大剂量应用时对癌细胞杀伤作用较为显著,但因其严重的毒副作用及耐药性而限制其广泛应用。目前有实验表明AKT异常升高可能与顺铂耐药性有关，但顺铂通过何种信号通路来调控AKT目前较少涉及,因此,在临床上克服应用顺铂治疗肿瘤产生的耐药,仍需要进一步开展大量系统性实验。实验一顺铂诱导ROS产生及顺铂联合ROS抑制剂NAC对结肠癌细胞增殖抑制与凋亡作用的研究目的：顺铂进入细胞后引起细胞内ROS异常增加,ROS在调控应激、细胞增殖、凋亡、信号通路活性等方面发挥重要作用。通过抑制ROS是否增加顺铂化疗敏感性作用机制目前仍不明确。本文的目的是验证顺铂是否产生ROS及抑制ROS是否具有增加顺铂敏感性作用。方法：以结肠癌细胞HCT-116为实验对象。设定ROS特异性抑制剂NAC的浓度20μmol/L、顺铂的浓度100μmol/L,两者合用处理结肠癌HCT-116细胞株。流式细胞术和免疫荧光检测顺铂产生的ROS; MTT法和流式细胞术检测顺铂(Ⅰ组)和NAC+顺铂(Ⅱ组)对结肠癌细胞株HCT-116生长及凋亡的影响,以验证NAC是否增加顺铂治疗效果。结果：顺铂可以导致细胞内ROS的异常积聚,顺铂联合NAC可以增强顺铂对结肠癌细胞HCT-116增殖的抑制及凋亡。结论：NAC可以增强顺铂化疗敏感性,本文研究结果为临床上为增加顺铂敏感性提供理论依据。实验二顺铂联合NAC调控AKT表达作用机制的研究目的：AKT在多种肿瘤细胞中成过表达状态,其主要发挥抗凋亡作用。然而,在顺铂处理的结肠癌细胞中AKT成过表达状态,顺铂通过何种途径激活AKT尚有待于进一步研究。本文在细胞水平上,观察顺铂对结肠癌细胞的作用,并对顺铂调控AKT表达及联合应用NAC是否影响AKT的表达；应用STAT3SiRNA和STAT3质粒转染结肠癌细胞检测对AKT表达的影响；以及应用染色质免疫沉淀检测STAT3在转录水平对AKT的影响。方法：分别用0、25μM、50μM、100μM及200μM顺铂,干预结肠癌细胞6h；设定顺铂浓度干预结肠癌细胞6h。应用STAT3SiRNA和STAT3质粒转染结肠癌细胞。]RT-PCR及Western-blot法检测AKT mRNA和蛋白水平的变化。以及应用染色质免疫沉淀检测STAT3在转录水平对AKT启动子的影响。结果：AKT的表达与顺铂浓度在一定范围内成正比,顺铂与NAC联合用药对降低STAT3, AKT的表达。转染STAT3SiRNA以及STAT3质粒的结肠癌细胞分别可以降低和增加AKT的表达；应用染色质免疫沉淀法发现检测STAT3与AKT启动子结合进而影响其转录水平的变化。结论：顺铂激活STAT3/AKT信号通路参与耐药的产生,而STAT3可以在蛋白水平与转录水平调控AKT的表达,应用NAC较显著的提高顺铂敏感性,100μM顺铂与20μM NAC合用可以达到较大的杀伤效果且产生耐药性的机率较小。实验三顺铂联合NAC在裸鼠体内对AKT表达的影响目的：研究顺铂联合NAC对人结肠癌细胞系HCT-116细胞形成的移植瘤中AKT表达的影响。方法：将BALB/C裸鼠随机平分为3组,分别皮下注射HCT-116细胞系,培养35天后,处死裸鼠取出皮下肿瘤,分别计算注射顺铂及顺铂联合NAC各组移植瘤的大小,以及采用免疫组织化学染色法检测AKT在各组移植瘤组织内的表达情况。结果：三组裸鼠均致瘤成功。CDDP+NAC细胞组移植瘤平均大小明显低于CDDP及空白对照组细胞组,差异有统计学意义(P<0.05)；免疫组化显示AKT在CDDP组移植瘤内表达水平明显高于在CDDP+NAC细胞组所形成的移植瘤内的表达水平。结论：NAC+CDDP下调AKT的表达,提示NAC可能参与调控AKT的表达,应用NAC可较显著的提高顺铂化疗敏感性。更多还原

【Abstract】 Cisplatin is a neutral, inorganic square planar complexes. It can reacted with DNA, inducing to produce the unique biological effects, then repair the damage of DNA to cell survival or activation of the apoptotic program irreversible, leading to cell apoptotic. However, the interaction of cisplatin with the DNA, mainly through a series of spontaneous hydration reaction, which relates with the water molecules to replace the process of the cis-chloro ligand. This single hydration form is recognized as a form of the highly reactive, but such form formation rate is often limited by many endogenous nucleophilic substances, such as:glutathione (GSH), methionine metallothionein, and protein. Therefore, when the cisplatin enters cells, it has the potential vulnerability or more of these substances, as well as other cellular components result cytoplasm inactivated.Cytotoxic chemotherapeutic drugs such as cisplatin, which mainly reacts with the purine bases in the DNA of the nucleophilic N7locus, thereby forming the composite form of DNA-protein and DNA-DNA chain and inter-chain cross-linking. However, there is evidence that the chain formed adducts can induced cytotoxic effects occurred. In addition, there are reports that found a similar chain crosslinking DACH-sulfate platinum complexes in cultured cells, and this complex is mainly for the difference between a different quantity and quality of DNA bases. Cisplatin resistant tumor cells cells provide new clinical ideas for the possible destruction of such cause. Since the DNA adduct formed chain can cause a lot of cisplatin-induced nuclear damage, therefore, the decision of the level and degree of the total platinum and DNA binding cytotoxicity. Although cisplatin affect DNA replication, but its influence there is no correlation between the generation of DNA synthesis and cell toxicity. Therefore, each recognition protein may start one or more specific biological signal source of stress, so that the DNA damage to exhibit seemingly unrelated biological effect, this replication and transcription of the with adduct cisplatin destruction process is consistent, but such biological effects do not necessarily lead to cell death directly. In terms of the for cisplatin role of cell survival and pro-apoptotic signal simultaneously activated cisplatin treatment, the relative intensity and/or duration of activation and integration of downstream signaling molecules, and ultimately produce different biological effects and resulting in generation of cisplatin resistance. Colorectal cancer is the more common malignancies in the clinical, its incidence ranks third, caused by a higher mortality rate. Today, the treatment of tumors constant development and updating, an endless stream of new anti-cancer drug cisplatin is still more commonly used chemotherapy drugs in clinical cancer cell-killing effect, especially when in its application dose more significant but because of its serious side effects and drug resistance, limit its wider application. The experiments show that the the AKT abnormal expression of increase may be associated with cisplatin resistance, cisplatin by what signaling pathways to regulate AKT expression is less involved.Part1cisplatin induce the ROS production and combination with NAC to research the inhibition and apoptosis ratio in human colon cancer cellsPurpose cisplatin enter into the cells resulted in the ROS increased at an abnormal level. ROS play an important role in the reaction in the regulation of oxidative stress, cell proliferation and apoptosis, regulate the activity of signaling pathways. However, the mechanism of inhibiting ROS how to increase the chemosensitivity of cisplatin remains unclear. The purpose of this paper is to verify cisplatin generation of ROS and inhibit ROS whether increased sensitivity of cisplatin.Methods the colon cancer cells(HCT-116) served as the experimental subjects. Set the ROS specific inhibitor of NAC concentration at20μmol/L, the cisplatin concentration at100μmol/L, combined them treatment of colon cancer HCT-116cell lines. ROS generated HCT-116detected by flow cytometry and immunofluorescence; MTT method and flow cytometry cisplatin (I group) and NAC combined with cisplatin (I group) to detected the growth and apoptosis of colon cancer cell line,and to verify the NAC to increase the chemosensitivity of cisplatin.Results Cisplatin can cause abnormal accumulation of intracellular ROS, cisplatin combined with NAC can enhance cisplatin inhibition and apoptosis in colon cancer cells HCT-116Conclusions NAC can enhance cisplatin chemotherapy sensitivity, the clinical results of this study provide a theoretical basis for increased sensitivity to cisplatin. Part2cisplatin combined with NAC regulated the role of AKT and activated the expression of AKT in colon cancersPurpose The level of AKT in a number of tumor cells, its mainly served as anti-apoptotic effects. However, with application cisplatin treatment in colon cancer cells AKT was also overexpressed, but cisplatin how to activated the AKT was still unknown. In this paper, observed at the cellular level, the role of cisplatin in colon cancer cells, and to study whether AKT expression, cisplatin and combined with NAC inhibited the expression of AKT; application STAT3SiRNA and STAT3plasmid transfected cells to detect the expression of AKT; application chromatin immunoprecipitation to detected the STAT3regulated the promoter of AKT at the transcriptional level.Method Setting the cisplatin concentration at0.25μM,50μM,100μM and200μM, and interfere with the colon cancer cells at6h; Using STAT3SiRNA and STAT3plasmid transfected colon cancer cells. RT-PCR and Western-blot assay detected the level of AKT. Application chromatin immunoprecipitation detect STAT3combined with AKT promoter at the transcriptional level.Results The expression levels of AKT cisplatin concentration is proportional to within a certain range, cisplatin and NAC combination of lowering of STAT3and AKT expression levels. STAT3SiRNA transfection of colon cancer cells can be reduced and increased expression levels of AKT and using the plasmids of STAT3produced the opposite effect. Application chromatin immunoprecipitation method detected STAT3binding with AKT thereby affecting at a transcriptional level.Conclusion Cisplatin activate STAT3/AKT signaling pathway involved in the development of resistance, The STAT3can regulate AKT expression at the protein level and transcriptional level, application of NAC can significantly improve the sensitivity to cisplatin,100μM cisplatin in combination with20μM NAC can achieve greatest effect and produce a low probability of resistancePart3Cisplatin combined with NAC in vivo study of colon cancer cells in vivo tumorigenicity Purpose To study the impact of cisplatin combined with NAC on human colon cancer cell line HCT-116cells formed tumor growth.Method BALB/C mice were randomly levels were divided into three groups, namely subcutaneous injection of HCT-116cell lines, cultured for30days, the mice were sacrificed to remove subcutaneous tumors were calculated injection of cisplatin and cisplatin combined with NAC tumor xenografts size and weight, as well as by immunohistochemical staining to detect the expression of AKT in transplanted tumor tissue.Results three groups of nude mice were tumorigenic success. CDDP+NAC cell group xenografts average size and of CDDP significantly lower than the control group cells group, the difference was statistically significant (P<0.05); Immunohistochemistry showed that AKT the CDDP group of transplanted tumor expression levels were significantly higher than in the CDDP+the xenografts are formed in the NAC cell group expression levels.Conclusion NAC lowered the expression of AKT, suggesting that NAC may be involved in the regulation of AKT expression.更多还原