【作者】 赵冀安；

【导师】 王士杰；

【作者基本信息】 河北医科大学 ， 外科学， 2014， 博士

【摘要】 肝细胞性肝癌位居世界恶性肿瘤发病率的第五位，是全球第三位的癌症死亡原因。我国是全球肝癌发病率最高的国家，肝癌为我国第二位恶性肿瘤致死病因。由于该病起病隐匿、进展迅速、早期就诊率低，60%-70%的患者在确诊时已处于肿瘤晚期，丧失了手术治疗的机会，只能进行姑息性的对症支持治疗。至今肿瘤转移及其调控的确切机制仍不清楚，而且无法在临床上采取有效的治疗措施，因此，揭示恶性肿瘤转移及其调控的确切分子机制已成为临床提高肿瘤治疗效果的迫切需要。联氨基姜黄素是姜黄素的一种衍生物，同姜黄素相比，联氨基姜黄素有更强的水溶性和稳定性，具有较高的细胞通透性、抗肿瘤活性、药理活性较好的生物利用度。JAKs/STATs信号转导通路的持续活化与肿瘤发生发展过程密切相关。STAT3作为JAKs/STATs转导通路的重要成员，是联系胞外信号与细胞应答的桥梁。STAT3参与调节肿瘤的增殖、生存、转移、血管生成、免疫应答等过程。JAK2/STAT3信号通路的激活，特别是STAT3的激活对肿瘤的发生、发展有着重要影响。本研究采用分子生物学技术和病理学对联氨基姜黄素进行研究，探讨其体内外抗肝癌作用和其作用机制，为最终应用于临床奠定理论和实验基础。第一部分联氨基姜黄素抑制人肝癌细胞HepG2增殖和促进其凋亡目的：本部分通过观察姜黄素衍生物——联氨基姜黄素（HZC）对人肝癌细胞HepG2增殖和凋亡的影响，探讨HZC抑制HepG2细胞增殖的机制。方法：将HepG2细胞用不同浓度的CUR(10、20、40μmol/L)和HZC(10、20、40μmol/L)分别孵育12、24及48h后进行MTT检测。本实验进一步研究HZC和CUR抑制HepG2细胞的诱导凋亡作用，用含有不同浓度CUR(10、20、40μmol/L)和HZC(10、20、40μmol/L)的培养基培养HepG2细胞48h，利用流式细胞法测定细胞凋亡率。以不相同浓度CUR和HZC(10、20、40μmol/L)分别刺激肝癌细胞株HepG224h，通过Western blot检测Bcl-2蛋白家族蛋白表达情况。结果：MTT结果显示CUR和HZC对肝癌细胞株HepG2的半抑制浓度（IC50）分别为25.43±2.86μmol/L和5.84±0.97μmol/L。CUR和HZC均可抑制人肝癌细胞HepG2的增殖，且呈时间、浓度依赖性（P＜0.05）。相同的浓度（40μmol/L）的CUR和HZC作用于HepG2细胞的不同时间（12、24及48h），HZC对HepG2细胞的抑制率均高于CUR（P＜0.05）。流式细胞法测定结果显示，同对照组相比，不同浓度的HZC和CUR处理组凋亡率增加，且HZC更为显著。当CUR和HZC处理浓度为40μmol/L，细胞凋亡水平最高，HZC更为显著。CUR和HZC均可抑制HepG2细胞中Bcl-2和Bcl-xl蛋白的表达，并可上调Bax蛋白的表达，且呈浓度相关性（P＜0.05）。其中对Bcl-2和Bcl-xl蛋白表达的抑制作用和对Bax蛋白表达的促进作用HZC均强于CUR（P＜0.05）。结论：CUR和HZC均可抑制人肝癌细胞HepG2的增殖和诱导HepG2细胞凋亡，其中HZC的抑制增殖作用和诱导凋亡作用均强于CUR。HZC对Bcl-2蛋白家族蛋白表达的影响作用强于CUR。第二部分联氨基姜黄素抑制JAK2/STAT3信号转导通路对人肝癌细胞HepG2的影响目的:探讨姜黄素衍生物——联氨基姜黄素（HZC）通过抑制JAK2/STAT3信号转导通路，探讨HZC对人肝癌细胞HepG2的影响及其相关机制。方法:以人肝癌细胞HepG2为研究对象，分DMSO对照组、姜黄素处理组和联氨基姜黄素处理组。将HepG2细胞用不同浓度的HZC和CUR(10、20、40μmol/L)分别孵育24h，应用Western blot检测上述不同处理组HepG2细胞JAK2/STAT3信号转导通路相关分子的变化，包括上游分子P-JAK2、JAK2、P-STAT3、STAT3以及下游分子蛋白表达水平。结果：Western blot检测结果显示，CUR和HZC能抑制P-JAK2和P-STAT3的蛋白表达，并调节其下游与肝癌细胞凋亡的相关分子蛋白表达水平。相同浓度CUR和HZC比较，HZC对肝癌HepG2细胞P-JAK2、P-STAT3、Bcl-2，Bcl-xl，Bax，Cyt c，Caspase-3和Caspase-9蛋白表达的影响更显著，差异有显著性（P<0.05或P<0.01）。结论：CUR和HZC均能显著降低JAK2和STAT3的活化水平并且影响Bcl-2, Bcl-xl, Bax, Cyt c, Caspase-3和Caspase-9表达。CUR和HZC通过抑制JAK2/STAT3细胞信号转导通路，调节其下游与肝癌细胞生长、凋亡的相关分子蛋白表达水平，从而抑制肿瘤细胞增殖，诱导其凋亡，起到抑制肿瘤的作用。与CUR相比，HZC是一种更为有效的肝癌抑制药物，具有良好的应用前景。第三部分联氨基姜黄素对二乙基亚硝胺诱导雄性大鼠肝癌发生的影响目的：通过动物实验研究姜黄素(CUR)和联氨基姜黄素（HZC）对二乙基亚硝胺（DEN）诱导的SD雄性大鼠肝癌发生、发展的影响，为应用于临床奠定理论和实验基础。方法：1大鼠肝癌模型制备及药物干预:选取体重为100-120g健康雄性SD大鼠120只。适应性生存一周后，根据实验要求随机分为6组，分别为组1（正常对照组）、组2为肝癌模型组（DEN组）、组3为姜黄素对照组（CUR组）、组4为姜黄素防护组（DEN+CUR组）、组5为联氨基姜黄素对照组（HZC组）、组6为联氨基姜黄素防护组（DEN+HZC组）。正常对照组以80mg/kg生理盐水腹腔注射，2次/周，12周后停药。CUR对照组和HZC对照组以80mg/kg分别给予CUR和HZC腹腔注射，2次/周，12周后停药。DEN组、DEN+CUR组和DEN+HZC组均给予50mg/kgDEN腹腔注射，2次/周，12周后停药。同时DEN+CUR组和DEN+HZC组以80mg/kg分别给予CUR和HZC腹腔注射，2次/周，12周后停药。2血清学指标测定：第24周实验结束处死健在大鼠前抽取其血液，用自动生化分析仪检测丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)、碱性磷酸酶(ALP)、Y-谷氨酞转肤酶(GGT)、总胆红素水平(TBL)肝功能指标。3肿瘤行为学观察：试验结束时通过麻醉方法处死各组健在大鼠后立刻将大鼠肝脏解剖出，并且计数其肿瘤数目，并且观察肿瘤形态。同时对大鼠体重和肝脏重量进行测量。4HE染色和免疫组化分析：留取肝组织固定包埋，制成石蜡切片。通过HE染色比较各组大鼠肝组织病理变化。用免疫组化的方法检测各组大鼠肝组织中增殖细胞核抗原（PCNA）的表达情况。结果：1发病率和存活率：至24周试验结束时，2组（DEN组）、4组（DEN+CUR组）、6组（DEN+HZC组）发病分别为30只、11只和6只，3个对照组均无发病大鼠。死亡大鼠分别为12只、5只和2只。DEN组和DEN+CUR组之间、DEN组和DEN+HZC组之间的发病率均具有统计学差异（均P<0.01）。三个对照组大鼠均无发病及死亡。DEN组和CUR+DEN组之间、DEN组和HZC+DEN组之间的存活率均具有统计学差异（P<0.05和P<0.01）。DEN+CUR组和DEN+HZC组之间存活率及发病率虽均无显著差别，但DEN+HZC组发病率、存活率均优于DEN+CUR组。2肝脏肿瘤：直径大于6mm记为癌结节。DEN组为17.17±1.08个，DEN+CUR组为13.36±2.17个，DEN+HZC组为11.83±3.90个。三个对照组大鼠均无发现癌结节。DEN组和DEN+CUR组之间、DEN组和DEN+HZC组之间的发病率均具有统计学差异（均P<0.05）。DEN+CUR组和DEN+HZC组之间癌结节计数无显著差别，但DEN+HZC组癌结节计数低于DEN+CUR组。3体重和肝脏重量：正常对照组的大鼠体重大于DEN组(P<0.01)。DEN组的体重、体重增量小于DEN+CUR组和DEN+HZC组，且均具有统计学差异（均p<0.01）。DEN组的肝脏相对和绝对重量高于DEN+CUR组和DEN+HZC组，且均具有统计学差异（均p<0.01）。正常对照组、CUR组和HZC组间大鼠生长情况相似，体重、体重增长、肝脏相对和绝对重量差异无显著性(P>0.05)。4肝功能指标：正常对照组、CUR组和HZC组各项肝功能指标正常，差异无显著性(P>0.05)。与正常对照组比较，DEN组大鼠的ALT、AST、ALP、GGT、TBL明显升高，差异有统计学意义(P<0.01)；DEN+CUR组和DEN+HZC组中各项肝功指标较DEN组降低，差异有统计学意义(P<0.01)，尤其是DEN+HZC组。5肝组织病理学和免疫组化染色：正常组的肝组织显示肝小叶有正常的结构；CUR组和HZC组显示出中央静脉和肝血窦充血；DEN组显示出癌灶；DEN+CUR组显示癌灶伴局灶性坏死；DEN+HZC组显示癌灶伴片状坏死。正常组无PCNA染色，DEN+CUR组和DEN+HZC组的PCNA阳性率也低于DEN组(P<0.01)。结论：1给予SD大鼠小剂量多次间断腹腔注射DEN，可以成功制作大鼠肝癌的动物模型，以模拟人体原发性肝癌的发生、发展过程，进行有关肝癌发生机理的研究和各种干预试验。2HZC对DEN诱发的雄性大鼠肝癌具有显著的化学预防作用，明显降低肝癌的发生率。此外HZC具有抑制DEN对大鼠肝脏的毒性作用，对肝脏起到具有保护作用。3HZC可以有效减轻DEN诱导大鼠肝癌病理学的改变，降低DEN诱导的SD大鼠肝癌组织中PCNA的表达，预示着有较好的预后。更多还原

【Abstract】 Hepatocellular carcinoma (HCC) is the fifth commonest malignancyworldwide and is the third most common cause of cancer-related death.Incidence rate of HCC has been the most in China, where is the secondleading cause of cancer death now. Due to the onset occult disease, rapidprogress, and ineffective treatment,60%-70%of patients are usuallydiagnosed with advanced cancer and not available for the surgical treatment.They have to suffer from a palliative symptomatic treatment finally. Until now,the metastatic mechanisms and the regulated mechanisms of tumors are notknown clearly, so it is very important for understanding the mechanisms ofinvasion and metastasis and searching effective approaches to prevent therecurrence and metastasis of HCC is of great importance.Hydrazinocurcumin (HZC) is the analogue of curcumin (CUR).Compared with CUR, HZC has greatly improved water solubility and stability,and has high cell permeability, anti-tumor activity, improved bioavailabilitywith more favorable pharmacological activity. Persistent activation of signaltransduction pathway of JAKs/STATs is associated with the occurrence anddevelopment of tumors. STAT3is the important member of transductionpathway which connects extracellular signal to cell response. STAT3involvedin the regulation of tumor proliferation, survival, metastasis, angiogenesis andimmune response processes. Activation of the signal pathway of JAK2/STAT3,especially the activation of STAT3has an important influence on theoccurrence and development of tumors. In our study, the use of molecularbiology techniques and pathology on HZC which investigates theanti-hepatocarcinoma effect in vivo and vitro and its mechanism is applied tothe theoretical and experimental basis for the final clinical application. Part I Hydrazinocurcumin inhibits proliferation of humanhepatoma cell HepG2and induces cell apoptosisObjective: To investigate the effect of Curcumin analogue(hydrazinocurcumin, HZC) on proliferation and apoptosis of human hepatomacell HepG2and elucidate its mechanism.Methods: Different curcumin (CUR) and hydrazinocurcumin (HZC)concentrations (10,20,40μmol/L) were cultured for12h,24h and48h toHepG2cells proliferation was detected by MTT. For further study theinhibition of induction of apoptosis by CUR and HZC, we carried out differentconcentrations of CUR and HZC to culture the HepG2cells for48h in order todetermine the cell apoptosis rate by flow cytometry method (FCM).Expression of Bcl-2protein family in HepG2cells under differentconcentrations of CUR and HZC (10、20、40μmol/L) for24h, we compared theintensity of protein expression by Western blot.Results: MTT assay showed the IC50of CUR was25.43±2.86μmol/Lcompared with5.84±0.97μmol/L of HZC in HepG2cells. Both CUR and HZCcould inhibit the proliferation of HepG2cells, with a time and dose-dependentmanner (P<0.05). The inhibitory rate of HZC was higher than that of CUR at40μmol/L at different time (12、24and48h)(P<0.05). Compared with thecontrol group, both drugs had the ability to induce apoptosis in HepG2cells,and HZC was more potent than CUR. The level of apoptosis was higher in40μmol/L of CUR and HZC, especially the latter. It is shown that the expressionof Bcl-2and Bcl-xl is down-regulated while the expression of Bax isup-regulated after treated with CUR and HZC in HepG2cells, with adose-dependent manner（P＜0.05）. The stronger inhibitor of the Bcl-2andBcl-xl and promotion of Bax was HZC while the weaker one was CUR (P＜0.05).Conclusion: Both CUR and HZC could inhibit the proliferation andinduce apoptosis of HepG2cells, especially HZC. Impact of expression ofBcl-2protein family in HepG2cells by HZC was better than CUR. Part II Hydrazinocurcumin inhibits effect of signal transductionpathway of JAK2/STAT3on human hepatoma cellHepG2Objective: To investigate the effect of curcumin analogue(hydrazinocurcumin, HZC) on human hepatoma cell HepG2by inhibiting theJAK2/STAT3signal transduction pathway and elucidate its relevantmechanisms.Methods: The human hepatoma cell HepG2were divided into3groups,including DMSO control group, curcumin (CUR) treatment group andhydrazinocurcumin (HZC) treatment group. Treated with differentconcentrations of CUR and HZC (10、20、40μmol/L) at24h in HepG2cells, tostudy the effect on JAK2/STAT3signaling tranSDuction pathway, includingthe expression of P-JAK2, JAK2, P-STAT3, STAT3and its downstream targetswhich were detected by Western blot analysis.Results: The Western blot analysis demonstrated that HZC and CURdecreased the expressions of P-JAK2, P-STAT3in HepG2cells and regulatedits downstream targets which contributed to induction apoptosis. Comparedwith the same dose of CUR, HZC showed stronger effect on proteinexpression in P-JAK2、P-STAT3、Bcl-2，Bcl-xl，Bax，Cyt c，Caspase-3andCaspase-9（P<0.05or P<0.01）.Conclusion: Both CUR and HZC could significantly inhibit the theactiviation of JAK2and STAT3protein and affect expression of Bcl-2, Bcl-xl,Bax, Cyt c, Caspase-3and Caspase-9. CUR and HZC regulated thedownstream targets which contributed to suppression of cell proliferation, aswell as induction cell apoptosis through block of JAK2/STAT3signalingtransduction pathway, so did inhibition of proliferation of HepG2cells,induction of apoptosis and prevention the tumour progress. HZC was morefavorable pharmacological activity than CUR and might have translationalpotential as effective cancer therapeutics or preventive agent for human. Part III Preventive effect of hydrazinocurcumin oncarcinogenesis of diethylnitrosamine-inducedhepatocarcinoma in male SD ratsObjective: We studied the effect of CUR or HZC on hepatocarcinomainduced by diethylnitrosamine (DEN) in male SD rat, which could providetheoretical and experimental basis for clinical application.Methods:1Model preparation and drug intervention of hepatocarcinoma:120healthy male SD rats weighting100–120g were provided. Rats wererandomised divided into six groups accordance with the requirements afterone week of acclimatisation. Group1(control non-treated group) wasadministered intraperitoneally (80mg/kg) physiological saline twice a weekfor12weeks. CUR in group3(control CUR-treated group) was administeredintraperitoneally (80mg/kg) twice a week for12weeks. HZC in group5(control HZC-treated group) was administered intraperitoneally (80mg/kg)twice a week for12weeks. Rats in group2(DEN-bearing non-treated group)were given intraperitoneal injection of DEN (50mg/kg) twice a week for12weeks. Rats in group4(DEN-bearing CUR-treated group) were administeredwith both DEN as in group2and CUR as in group3. Rats in group6(DEN-bearing HZC-treated group) were administered with both DEN as ingroup2and HZC as in group5.2Determination of serum markers: The experiment was terminated at theend of24weeks and all the surviving living rats were sacrificed at the end ofthe experiment. Blood samples were taken before sacrifice. Determination ofserum alanine aminotransferase(ALT), aspartate aminotransferase(AST),alkaline phosphatase(ALP), γ-glutamyltransferase (GGT), total bilirubinlevel(TBL) were measured by automatic biochemical analyzer.3Tumor behavior observation: The surviving living rats wereanaesthetized and sacrificed at the end of the experiment. Liver was excisedimmediately and then counted tumors, observe tumor morphology. Meanwhilebody and liver weight were measured. 4HE staining and immunohistochemical analysis: Specimens were takenfrom the liver of rats. All the liver tissues were fixed routinely, and embeddedin paraffin. Comparison of pathological changes in liver tissues of each groupby haematoxylin and eosin (HE) staining. The expression of proliferating cellnuclear antigen (PCNA) in liver tissues of each group were detected byimmunohistochemical analysis.Results:1Incidence rate and Survival rate: There have47rats withhepatocarcinoma in all groups,30rats in group2(DEN-bearing non-treatedgroup),11in group4(DEN-bearing CUR-treated group),6in group6(DEN-bearing HZC-treated group),0in three control groups.12rats,5ratsand2rats were dead in group2, group4, group6, respectively. It showsSTATistical difference between group2and group4、group2and group6ininciDENce rate and survival rate (all. P<0.05). ALThough inciDENce rate andsurvival rate in group4and group6had no STATistical differences, the lattergroup was better.2Liver tumors: Nodule larger than6mm in diameter was considered ascancerous node. In rats of group2, the average count of nodule was17.17±1.08, while it was only13.36±2.17,11.83±3.90in the group4and group6,respectively. No hepatic nodule was observed in three control groups.although there was no statistical significance between group4and group6,they all declined count of nodules significantly compared with group2,especially group6.3Body weight and liver weight: The body weight in rats of group1werelarger than group2. Body weight and gain body weight in group2weresignificantly lower than group4and group6（all. p<0.01）. We measured theabsolute and relative liver weight which was significantly higher in group4orgroup6than in group2（all. p<0.01）. There were no differences in bodyweight, absolute and relative liver weight in three control groups (P>0.05).4Liver function index: The index of liver function in control groups werenormal. Rats from group2showed an significant increase in liver enzymesactivities in comparison to group1(P<0.01). The serum from rats in group2 showed a highly significant increase in levels of liver enzymes whencompared to those from group4or group6, especially the latter group(P<0.01).5Liver histopathology and immunohistochemical staining: Hepatictissues from the group1showed hepatic lobules with normal architecture;Hepatic tissues from group2and group5indicated moderate to severe venousand sinusoidal congestion; Hepatic tissues from rats in group2showed cancerfocus; Hepatic tissues from group4showed cancerous focus with focalnecrosis; Hepatic tissues from group6showed cancerous focus with patchynecrosis. Immunohistochemical staining in group1showed no PCNA stainingpattern. The positive tumor for PCNA was significantly lower in the group4and group6than in group2(all. P<0.05).Conclusion:1The course of hepatocarcinoma rat model induced by alow dose of DEN discontinuously is similar with the generating occurrenceand development of human primary hepatocarcinoma. So it is convenient forus to carry out the research and intervention test in mechanism ofhepatocarcinogenesis.2HZC had significantly preventive effect on carcinogenesis ofDEN-induced hepatocarcinoma in male SD rats and decreased the incidenceof hepatocarcinoma. Moreover administration of HZC had a protective effectagainst toxicity of the liver induced by DEN.3HZC could effectively reduce DEN-induced pathological changes inhepatocarcinoma of SD rats and decreased the expression of PCNA inhepatocarcinoma tissues, indicated a better prognosis.更多还原