【作者】 吴爽；

【导师】 张阳；

【作者基本信息】 大连医科大学 ， 肿瘤内科学， 2010， 硕士

【摘要】 背景：原发性肝细胞癌(hepatocellular carcinoma,以下简称肝癌)是目前最常见的恶性肿瘤之一,发病率和死亡率逐年上升。传统化疗仍是晚期肝癌主要的治疗手段,但多数患者合并有失代偿性肝硬化、肝功能异常,难以耐受常规剂量化疗,是目前肝癌治疗失败、患者无法临床获益的主要原因。因此,寻找更加高效、低毒的治疗模式以提高总体疗效成为目前肝癌治疗研究的重点。分子靶向药物—索拉非尼(Sorafenib)具有直接抑制肿瘤增殖和阻断肿瘤新生血管形成的双重抗肿瘤的作用,使得肝癌治疗效果有所改善；节拍式化疗(metronomic chemotherapy)以血管内皮细胞为作用靶点,与传统化疗相比,具有患者依从性高、耐药现象减少、免疫功能增强、毒副反应小等明显优势。两者联合,作用机制不同,理论上具有协同增效作用。前期实验已证实新一代抗肿瘤药物伊立替康(CPT-11)对肝癌治疗有效,细胞毒作用强于临床常规用药5-FU、DDP。因此本实验选择将Sorafenib与伊立替康(CPT-11)节拍式化疗联合作用于小鼠H22肝癌移植瘤模型,进行疗效评价及基础性研究,为其可能的临床应用提供实验依据。目的：观察伊立替康(CPT-11)节拍式化疗联合索拉非尼对小鼠移植瘤生长的抑制作用,通过检测血管内皮生长因子(VEGF)及微血管密度(MVD)的表达探讨其可能的相关机制。方法：1.建立小鼠H22肝癌移植瘤模型。2.随机分组：空白对照组(A)：0.9%生理盐水腹腔注射0.2ml,qd,(d1-28)+0.9%生理盐水胃管灌入0.2ml,qd,(d1-20)；Sorafenib组(B)：0.9%生理盐水腹腔注射0.2ml,qd,(d1-28)+索拉非尼50mg／Kg,胃管灌入0.2ml,qd,(d1.-20)：CPT-11节拍式给药(LDM)组(C)：CPT-114mg／Kg腹腔注射0.2ml,qd(d1·28)+0.9%生理盐水胃管灌入0.2ml,qd,(d1-20)；CPT-11常规剂量(TDM)组(D)：CPT-1175mg／Kg腹腔注射0.2ml,2次／W,(d1-28)；Sorafenib+CPT-11(LDM)组(E)：CPT-114mg／Kg腹腔注射0.2ml,qd,(d1-28)+索拉非尼50mg／Kg,胃管灌入0.2ml,qd,(d1-20)。3.停药24小时后处死小鼠,测量瘤体积及瘤重,计算抑瘤率,并用免疫组化SP法测定肿瘤组织中的VEGF和CD31的表达。结果：1.索拉非尼、CPT-11对小鼠H22肝癌的生长均有抑制作用,用药后各实验组肿瘤体积均小于对照组；2.A、B、C、D、E各组用药后平均瘤重分别为：5.20g、3.53g、3.54g、3.63g、2.34g,统计学分析后,除B、C、D组间无差异外,其余组间比较均有差异,P均<0.05。与对照组相比,以联合用药组肿瘤负荷最低；B、C、D、E组相应抑瘤率分别为：32.01%、31.86%、30.14%、54.9%。各组抑瘤率比较,AB、AC、AD组间无差异,而E组抑瘤率明显高于其他四组,AE组比较P<0.01；3.小鼠一般状态观察：A、E组小鼠出现进食饮水减少、精神萎靡、体重下降等症状较其他实验组更为明显,并且E组小鼠出现明显腹泻；4.HE染色：A组大部分肿瘤细胞生长旺盛,核分裂相多见,只有少量细胞出现坏死。B、C、D、E组瘤体内坏死组织多,多数肿瘤细胞有细胞核结构不清、核固缩、核碎裂及核溶解现象,残存细胞核分裂减少,以E组肿瘤细胞坏死最为明显；5.免疫组化检测VEGF,与A组相比,B、C、E组VEGF表达显著减少,(P均<0.05),其中以E组最为明显。AD组间无明显差异；6.免疫组化检测CD31,高倍镜下,A、D组可见大量染成棕黄色的血管内皮,而B、C、E组被着色的血管内皮较少。计数各组MVD值并做组间比较,AB、AC、AE组间差异显著,P均<0.05,AD间无差异。BE、CE组间差异显著,P均<0.01。结论：1.索拉非尼和伊立替康节拍式化疗均可抑制小鼠H22肝癌移植瘤生长,联合用药抑制作用更强,表现为协同效应。2.两药联合能够协同抑制VEGF、MVD表达,抑制肿瘤血管形成3.两药联合不增加药物的毒副作用。更多还原

【Abstract】 Background:HCC (hepatocellular carcinoma) is one of the most common malignant tumor in worldwide,and medical chemotherapy still is a major treatment in advanced hepatocellular carcinoma. Most patients associated with decompensated cirrhosis and liver function deterioration,all of these had increased the difficulty of HCC treatment and leads to the failure of chemotherapy.Therefore,searching for a new way with more efficient and low toxicity to improve the overall efficacy of the HCC treatment had became the current focus.The emergence of molecular targeted drugs-Sorafenib give hope to the treatment of HCC,but the overall effect was unsatisfactory.The rapid proliferation of vascular endothelial cells were viewed as the target of metronomic chemotherapy.Compared with traditional chemotherapy,it has many advantages,such as:high compliance of patients, reduction of drug resistance,enhancing immune function, little toxicity,et al.These two methods have different mechanisms,so the combination of them may has synergism in theory.In the other hand, Irinotecan(CPT-11) has been confirmed that it has the effect of HCC treatment,and the cytotoxicity is stronger than 5-Fu and DDP in the early experiments.So in this study,Sorafenib combined with metronomic Irinotecan(CPT-11) chemotherapy were used on H22 liver cancer tumor-bearing mice,confirmed that the two combined had a synergistic anti-tumor effect and provided experimental evidengce for possible clinical application.It was.significant for improving the overall efficacy of the HCC treatment. Objective:To observe the inhibition effects of low dose CPT-11 Combined with Sorafenib on the growth of tumor transplanted from a mouse and its effect on the expression of vascular endothecial growth factor(VEGF) and microvessel density(MVD).Methods:1. mouse tumor model was set up by implanting H22 mouse liver cancer cells under the skins of mouse.2. Seventy-five experimental mice were randomly divided into five groups:control group(A),Sorafenib-treated group(B),CPT-11(LDM)-treated group(C),CPT-11(TDM)-treated group(D) and combination-treated group(E). Evaluate volume of tumor in five groups before and after treatment.3. Execute the mice at the twenty-eight day after treatments,to strip and weigh the neoplasm for evaluating the inhibition of the anti-tumor. Determine tumor tissue VEGF protein expressions and micro-vessel density(MVD),(assessed by CD31 immunohistoehemistry).Results:1. Both Sorafenib and CPT-11 affected the growth of tumor,The mean tumor volume of control group is the largest one in all groups after treatments.2. The mean tumor quality was 5.20g,3.53g,3.54g,3.63g,2.34g and the inhibition rate was 32.01%,31.86%,30.14%,54.9%in B,C,D,E respectively. The combination group had lower tumor burden and higher inhibition rate than the other groups.3. The mice of A,E appear to reduce the consumption of drinking and food, low spirit,weight loss more obvious than other groups.Even there were significant diarrhea in mice of E.4. HE staining:Most tumor cells of A group had vigorous growth and mitotic more common,only a few cells were necrotic.In the other groups, most tumor tissue were necrotic,and the phenomenon of nuclear condens-ation,nuclear fragmentation and nuclear dissolution were more common, especially in E group.5.Immunohistochemical detection of VEGF:compared with A,B,C,D groups significantly red(?)ced VEGF expression,(P all<0.05),of which the most significant E group.No significant difference between the AD group;6.Immunohistochemical detection of CD31:In high magnification,Aand Dgroup showed a lot of dyed brown vascular endothelium,while B,C,E groups were less colored vascular endothelial.Counted MVD in each group, AB,AC,AE,BE,CE had significant differences between groups,while AD had no difference.Conclusion:Metronomic CPT-11 chemotherpy combined with Sorafenib have significant inhibitory effect on the growth of the tumors and the expression of the VEGF and MVD in H22 liver cancer tumor-bearing mice.Metronomic chemotherpy combined with molecularly targeted therapy may be a new way of anti-tumors.更多还原