【作者】 朱冰；

【导师】 刘耕陶；

【作者基本信息】 中国协和医科大学 ， 药理学， 1996， 博士

【摘要】 原发性肝癌的发生除与病毒性肝炎有着密切的关系外，与致癌物对肝脏的损伤亦有关系如果治疗肝炎的药物同时具有阻断肝脏痛变发生的化学预防作用，将具有重要的意义，另外，肿瘤细胞的耐药性是临床肿瘤治疗的一大障碍，原发性和获得性耐药使肿瘤的的化学治疗失效，增大抗癌药物的用量有时又可引起骨髓、心脏和肝脏等器官的毒性，如果治肝炎的药物同时可以增加耐药肿瘤细胞对化疗药物的敏感性，特别是肝癌，将有助于提高临床抗肿瘤药物的化疗效果和降低因耐药而引起的毒、副作用，中性粒细胞参与急慢性肝炎的肝脏炎症性病理损伤过程，抗肝炎药物如能抑制中性粒细胞对肝细胞的损伤，将有利于肝细胞的修复。SY-801是我所研制的新一代抗肝炎药物，对化学性、免疫性肝损伤具有显著的保护作用，而且具有抗乙肝病毒、抗肿瘤和一定的诱导癌细胞分化的作用，在此基础上，本文进一步研究了SY-801在癌变的化学预防、肿瘤细胞耐药的逆转以及中性粒细胞所致肝细胞损伤保护等方而的作用和相关的机制，为SY-801在临床中的用途作进一步的理论探讨，本研究的主要内容包括以下三个方面：1．癌变的化学预防作用体外实验中采用3-甲基胆蒽和TPA二阶段联合转化小鼠Balb／3T3细胞实验模型，发现SY-801(10^-6～10^-4M)对3T3细胞恶性转化具有明显的抑制作用，使转化灶数目明显减少、转化细胞的恶性程度明显下降，在10^-6M浓度下，其作用强度与已知的癌化学预防药物维甲酸（RA）相当。3T3细胞增殖实验和Dot-blot分析表明，SY-801对TPA诱发的静止态Balb／3T3细胞³H-TdR掺人和细胞数目的增加具有明显的抑制作用，并对TPA诱导的H-ras、c-myc和PKCα基因mRNA表达升高具有明显的抑制作用、而对P53基因的表达具有显著的增强作用。体内实验表明，全程灌胃给SY-801（200，100mg／kg／day），对二乙基亚硝胺（DEN）诱导的大鼠肝癌前病变具有显著的抑制作用，可降低癌前病变肝组织中γ-GT阳性灶的数密度和而密度，降低肝胞浆液中GST-π蛋白表达的水平以及GST总酶活性和GSH量，降低肝胞浆液和微粒体中甲胎蛋白（AFP）的含量，Northern-blot和Dot-blot分析表明，SY-801对癌前病变肝组织中N-ras、c-myc、PKCα和GST-π基因mRNA的表达升高具有抑制作用，而对P53基因的表达则有增强作用，Western-blot分析表明，SY-801还具有抑制多药耐药药基因产物Mdr-1蛋白在癌前病变肝组织中表达增强的作用，以上结果表明，SY-801能够防止DEN诱发大鼠肝组织中的一系列生化特征和基因表达向恶性化、低分化和高增殖的癌变方向发展，起到化学预防的作用。为进一步了解SY-801在体内阻断DEN诱发肝癌前病变的作用与DEN代谢突径的关系，又研究了体内给SY-801对大鼠肝脏微粒体在体外代谢DEN作用的影响；同时，还研究了SY-801对DEN诱发肝细胞DNA损伤的作用和体外抗氧化作用，正常大鼠连续灌胃给SY-801三天，剂量200，100mg／kg／天，发现SY-801在诱导大鼠肝微粒体P-450总酶含量增加的同时，肝微粒体P-450代谢DEN的水平明显加强，表现在DEN脱乙基化和脱硝基化的产物均明显增加，尤其以解毒的脱硝基化途径为明显，而SY-801对与DEN结构十分类似的二甲基亚硝胺（DMN）经大鼠肝微粒体的代谢却没有影响。同样条件下，SY-801还显著诱导正常大鼠肝胞浆液中的GST活性，并以DCNB为底物的μ亚型活性升高最为显明；而Western-blot分析表明，SY-801对正常大鼠GST-π的极低表达没有影响，同时，SY-801对于DEN诱发体外培养大鼠肝细胞DNA损伤而导致的非程序DNA合成（UDS）的升高具有显著的抑制作用，表明预先给SY-801可增加DEN在正常大鼠肝脏中的解毒代谢和灭活后排出体外，防止肝脏及其它组织的DNA损伤，另外，SY-801还具有抑制黄嘌呤氧化酶系统生成O_2^-和Fenton反应生成OH·的作用。2．对肿瘤细胞耐药的逆转作用本实验以体外培养的耐受长春新碱（VCR）的人口腔癌KBv200细胞和耐受阿霉素（Adr）的人乳腺癌Adr^R MCF-7细胞为模型，探讨了SY-801对肿瘤细胞耐药性的影响及其相关机制，结果表明，给SY-801(2.5×10^-5～1×10^-4 M)可明显逆转上述细胞的耐药性，其中以逆转KBv200细胞耐受VCR的作用最强，1×10^-4、5×10^-5和2.5×10^-5 M SY-801分别逆转20.7、7.3和2.8倍；对于KBv200细胞交叉耐受紫杉醇（Taxol）和Adr^R MCF-7细胞耐受Adr，SY-801亦表现出显著的逆转作用，10^-4M时可分别逆转13.4和11.1倍。SY-801同时能够增强VCR对不耐药的敏感株KB细胞的杀伤作用，而对Taxol对KB细胞以及Adr对敏感株MCF-7细胞的杀伤作用均无影响。耐药的肿瘤细胞最显著的特征是抗肿瘤药物在细胞内的蓄积下降，耐药的Adr^R MCF-7细胞与敏感株MCF-7细胞相比，细胞内的Adr浓度只能达到后者的1／5，而经SY-801预先处理24～72小时或给Adr同时给SY-801，Adr在Adr^R MCF-7细胞内的蓄积浓度均有显著的提高。耐药肿瘤细胞内多药耐药基因编码的细胞膜P-糖蛋白表达的增加是其耐药的最主要机制。P-糖蛋白通过泵出抗肿瘤药物而使细胞内的蓄积下降；Western-blot分析表明，KBv200和Adr^R MCF-7细胞经SY-801处理12～72小时后，可显著降低耐药细胞内Mdr-1蛋白的高表达，免疫组织化学分析证实，上述细胞经SY-801处理后，细胞膜上Mdr-1蛋白的表达明显减少。GSH和GST与肿瘤的耐药亦密切相关，KBv200和Adr^R MCF-7细胞内的GSH含量均比敏感株的高出一倍左右，Adr^R MCF-7细胞内的GST活性是MCF-7的17.4倍；经SY-801处理后，上述两种耐药细胞内的GSH含量均明显降低，Adr^R MCF-7细胞内的GST活性亦明显降低。Bcl-2基因是细胞内控制死亡过程的重要基因之一，它可以抑制抗肿瘤药物引起的细胞毒作用及程序化死亡（apoptosis），包括Adr^R MCF-7细胞在内的许多耐药肿瘤细胞内的表达明显增高；Western-blot分析表明，经SY-801处理后的Adr^R MCF-7细胞内的Bcl-2蛋白的表达显著降低。3．对中性粒细胞、过氧化氢所致原代培养肝细胞损伤的保护作用及对中性粒细胞功能的影响活化的中性粒细胞（PMNs）在体内急性和慢性肝脏炎症反应起着重要的作用，其过量释放的氧自由基类还可能参与致癌和促癌过程。实验表明，SY-801对于A23187刺激PMNs所致的原代培养大鼠肝细胞的损伤具有保护作用，可以减少GPT的释放和肝细胞因损伤而引起的形态学上的改变。过氧化氢（H₂O₂）是活化的PMNs等免疫和炎症参与细胞所释放的一类重要的炎症介质，部分介导了PMNs引起的肝细胞损伤；给SY-801可显著抑制H₂O₂引起肝细胞损伤所造成的GPT释放增加和因膜脂质过氧化所造成的细胞膜MDA生成增加和膜流动性下降，扫描电镜观察亦表明SY-801对肝细胞的损伤有保护作用。通过研究SY-801对于大鼠腹腔PMNs经刺激活化的影响发现，SY-801具有显著抑制FMLP、PMA、F-离子、PMA+A23187四种不同刺激剂引起的PMNs释放O^2-，以及抑制PMA刺激PMNs释放H₂O₂；同时，SY-801对FMLP和A23187刺激PMNs活化时[Ca²⁺]i的升高有抑制作用，而[Ca²⁺]i的升高介导了PMNs脱颗粒、炎症介质释放等多种功能。表明SY-801既通过直接降低H₂O₂的细胞毒性又通过抑制PMNs活化以及释放氧自由基类等炎症介质来实现减轻PMNs所致肝细胞的损伤。综上所述，本文应用多种方法，从多层次研究了SY-801在癌化学预防、肿瘤细胞耐药逆转和肝细胞保护几方面的作用及其相关的生化、分子机理，首次发现肝保护剂SY-801对癌变有化学预防作用，又首次发现非钙拮抗剂的具有细胞保护和解毒作用的SY-801亦具有逆转肿瘤细胞耐药的作用，这些为SY-801用于与肝炎密切相关疾病-肝癌的预防，以及用于克服肿瘤化疗耐药和降低化疗药物的毒性提供了新的实验依据，对评估SY-801临床应用的前景具有重要的理论和实际意义。更多还原

【Abstract】 It is known that the occurrence of primary hepatocarcinoma is closely related to viral hepatitis as well as hepatocytes injuries induced by carcinogens. If an anti-hepatitis drug has blocking or suppressing effects on the development of hepato-carcinogenesis, it would be of great value. In addition, the emergence of drug resistance of tumor cells is a major problem in cancer chemotherapy. The intrinsic and acquired resistance of carcinoma to anti-cancer agents could cause failures of chemotherapy or toxicities of many organs such as bone marrow, heart, liver, as the dosage of anti-tumor drugs increased. If a hepatoprotectant possesses activity of increasing the sensitivity of carcinoma to chemotherapy, it will improve the efficiency of chemotherapeutics and reduce their toxic and side effects. Polymorphonuclear neutrophils （ PMNs ） participate in the inflammatory lesions of chronic hepatitis. If an anti-hepatitis drug could prevent the PMNs-induced injury to hepatocytes it should contribute to the reparation of liver injuries. SY-801, a new hepatoprotectant developed by our institute, showed significantly protective action against chemical and immunological hepatic injuries in mice and rats. It also has activities of anti - duck hepatitis virus B, anti-tumor and induction of cancer cells differentiation. On the bases of these investigations, the actions of SY-801 in chemoprevention of carcinogenesis, reversal of drug resistance and protection against hepatocyte injury as well as their related mechanisms were studied in the present study so as to further theoretically explore the clinical applications of SY-801. The results were summarized in three aspects as follows:1. Chmopreventive effect on carcinogenesisThe Balb / 3T3 cells were two stage transformed by co-use of 3-methylcholanthrene （ 3-MC ） and TPA in vitro. The results showed that SY-801 at concentrations of 10^-6 - 10^-4 M could obviously inhibit the malignancy of Balb / 3T3 cells expressed in decrease of transformed foci and reduction of malignant degree of transformed cells. The potency of SY-801 to inhibit such transformation was equal to the known positive cancer chemopreventive drug all trans-retinoic acid （ RA ） at the same concentration of 10^-6 M. The experiments of cell proliferation show that SY-801 obviously inhibited the increase of cell numbers and incorporation of ³H-TdR into quiescent Balb / 3T3 cells which induced by 100 ng / ml TPA with low concentration of serum （ 2.5 % ） or 2.5μg / ml insulin in serum-free culture medium. Dot blot assay of mRNA indicated that SY-801 suppressed the TPA-induced overexpression of H-ras, c-myc and PKCαgenes, whereas, markedly improved the expression of P53.In vivo, oral administration of SY-801 （100, 200mg/kg/day） markedly suppressed diethylnitrosamine （DEN）-induced praneoplasitic lesions in rat livers. Both number and area density ofγ-glutamyltransferase positive （γ-GT⁺） foci in preneoplastic livers were obviously reduced in SY-801 treated rats. It was also observed that significant decreases of mRNA and protein expression of GST-πand total GST activity as well as cytosolic GSH andα-fetal protein in SY-801 treated rats. Northern blot and dot blot assay showed that SY-801 reduced the overexpression of N-ras, c-myc and PKCαmRNA in the preneoplatic livers, whereas, P53 expression was enhanced. The Western-blot indicated that SY-801 could also inhibit the Mdr-1 protein expression of multiple drug resistance gene in DEN-induced preneoplastic livers. All the results suggested that the chemoprevantive effect of SY-801 relied on its inhibition of biochemical characteristic alternations and oncogene expressions in malignant transformation, low differentiation and hyperplastic hepatic lesions induced by DEN.In order to know the relation of DEN metabolism with its induced hepatic praneoplastic lesion, the metabolism of DEN in vitro by the hepatic microsomes from rats treated with SY-801 for three days was studied. The results indicated that the metabolism of DEN by rat hepatic microsomes was obviously enhanced as accompanied with significantly increase of hepatic microsomal P-450 content. Deethylation and particular denitrosation pathways of DEN metabolism by the hepatic microsomes from SY-801 treated rats were both enhanced. However, SY-801 showed no effect on dimethylnitrosamines （DMN） metabolism in the same conditions, although the chemical structure of DMN is similar to DEN. The increase of cytosolic and microsomal GSTs activities in SY-801 treated rat liver were also observed, especially for the GSTμclass isoenzyme as using DCNB for substrate. However, no expression of GST-πby Western-blot assay in normal and SY-801 treated rat livers was found. The DNA damage and its associated unscheduled DNA synthesis （UDS） in DEN treated rat hepatocytes was also observed in vitro. Co-incubation of SY-801 for 4 and 18 hr showed inhibitory effect on UDS of rat hepatocytes induced by DEN. All the results suggest that SY-801 could increase the detoxication and elimination of DEN in inactivating forms, which result in preventing DNA damage in rat livers. In addition, SY-801 inhibited the superoxide anion （O₂^-） and hydroxyl free radicals production in Xanthane oxidase system and Fenton reaction, respectively. 2. Reversal effect on drug resistance of carcinomaThe effects of SY-801 on drug resistance of two carcinoma cell lines: vincristine （VCR）- resistant human stomatic epidermoid carcinoma KBv₂₀₀ and adriamycin （Adr）-resistant human breast carcinoma Adr^R MCF-7 and their related mechanism were investigated in vitro. The results indicated that SY-801 (2.5×10^-5～1×10^-4 M) obviously reversed the drug resistance of the two kinds of tumor cell lines in dose-dependent manner. The reversal rate of SY-801 at the concentrations of 1×10^-4, 5×10^-5 and 2.5×10^-5 M on KBv₂₀₀ resistant to VCR was 20.7, 7.3 and 2.8 folds, respectively, as compared with the untreated group. 10^-4 M SY-801 also showed reversal effect on KBv₂₀₀ cross-resistant to anti-tumor drug Taxol and Adr^R MCF-7 resistant to Adr for 13.4 and 11.1 folds, respectively. In addition, SY-801 enhanced the cytotoxic effect of VCR against sensitive KB cell line for about several times, whereas no effect on Taxol to sensitive KB cells and Adr to sensitive MCF-7 cells was demonstrated.The decrease of antitumor drugs accumulation in cells was the most outstanding characteristic of drug resistant carcinoma cell lines. It was found that the intracellular concentration of adriamycin in resistant Adr^R MCF-7 cells was just about one fifth of the sensitive MCF-7 cells. Pretreatment with SY-801 for 24-72 hr or simultaneous coincubation with SY-801 obviously elevated the Adr accumulation in Adr^R MCF-7 cells. The major mechanism for carcinoma drug resistance was postulated the reason of overexpression of P-glycoprotein in cell membrane which encode by multiple drug resistant genes （MDR）. Western-blot assay indicated that treatment of KBv₂₀₀ and Adr^R MCF-7 cells with SY-801 for 12～72 hr, the Mdr-1 protein in cell membrane was markedly reduced. Immunohistochemical assay also confirmed such results. The role of GSH and GST in carcinoma drug resistance was attached importantly in recent years. The GSH content in KBv₂₀₀ and Adr^R MCF-7 cells were higher than their sensitive cells for almost about 1 fold, and the GST activity in Adr^R MCF-7 cells was about 17.4 folds as compared with MCF-7 cells. It was observed that both GSH content in KBv₂₀₀ and Adr^R MCF-7 cells as well as GST activities in Adr^R MCF-7 cells were markedly decreased after these cells were treated with SY-801 (1×10^-4 M) for 24～72 hr. Bcl-2 gene plays an important role in regulation of cell death. It could suppress the toxicity and apoptosis induced by antitumor drugs. Many drug resistant carcinoma including Adr^R MCF-7 cells show overexpression of Bcl-2 as compared with sensitive cell lines. SY-801 obviously reduced the elevation of Bcl-2 expression in Adr^R MCF-7 cells by Western blot assay. 3. Protection against polymorphornuclear neutrophils （PMNs） and hydrogen peroxide （H₂O₂） induced injuries of primary cultured rat hepatocytes, and effect on the function of PMNs.Activated PMNs show important role in acute and chronic liver inflammatory lesions. The over production of oxygen Free radicals and other inflammatory mediators from PMNs also involve in the processes of mutation and promotion of carcinogenesis. The results indicated that SY-801 protected the primary cultured rat hepatocytes from the cytotoxicity induced by A23187-stimulated rat peritoneal PMNs expressed in reduction of GPT release and morphological damages of rat hepatocytes. H₂O₂ is an important inflammatory mediator in immune and inflammatory participated cells such as PMNs. It partially mediates the cytotoxicity of PMNs toward hepatocytes. SY-801 obviously inhibited the H₂O_2- induced hepatocyte injury and lipid peroxidation in cell membrane as GPT release and MDA accumulation reduced and the cell membrane fluidity increased. The scan electromicrscopy indicated the damage of rat hepatocytes surface induced by H₂O₂, while, SY-801 showed protective effects on this damages.In addition, SY-801 influenced the activating processes and release of inflammatory mediators such as oxygen free radicals （OFRs） by PMNs. SY-801 markedly reduced the O^2- production by activating PMNs which challenged by four different kinds of stimuli FMLP、PMA、fluoride ion and PMA+A23187. The H₂O₂ production by PMA-stimulated PMNs was also inhibited. The elevation of intracellular calcium concentration of PMNs modulates many functions of PMNs such as the degranulation and OFRs production. SY-801 inhibited FMLP and A23187 induced [Ca²⁺]i elevation. All the results indicated that the protective effect of SY-801 on the PMNs induced hepatocytes injuries may through directly reducing the toxicity of H₂O₂ and suppressing the activation of PMNs and its associated over production of OFRs.In summary, it was the first time demonstrated that SY-801 with very low toxicity in vivo has actions in prevention of carcinogenesis, reversal of drug resistance and protection against hepatocyte injury mediated by PMNs. The related biochemical and molecular mechanisms of the actions of SY-801 was also elucidated in different ways and levels. The results of present study not only provide a new scientific basis for using SY-801 as a potential drug to prevent heaptocarcinogenesis and overcome drug resistance of carcinoma, but also have importantly theoretical and practical values in evaluating the clinical prospects of SY-801 in the long-term treatment of chronic viral hepatitis in the future.更多还原