【作者】 唐利；

【导师】 李雁；

【作者基本信息】 武汉大学 ， 肿瘤学， 2014， 博士

【摘要】 胃癌在内的腹盆腔恶性肿瘤易发生局域性进展,往往导致腹膜转移癌(peritonealcarcinomatosis, PC)。胃癌PC预后极差,其中位生存期约6个月。针对胃癌PC的传统治疗手段是以胃癌根治术为主,辅以全身化疗的综合治疗。然而,单纯手术仅切除肉眼可见肿瘤组织,无法清除腹腔内残余癌细胞[4-6]。因此,传统治疗方法不能有效延长PC患者的生存期。细胞减灭术(cytoreductive surgery, CRS)加术中腹腔热灌注化疗(hyperthermicintraperitoneal chemotherapy,HIPEC)在国际上探索了近30年,首先通过CRS切除肉眼可见瘤灶,再通过术中HIPEC清除腹腔内残余微癌灶和游离癌细胞,集根治手术、区域性化疗、热疗和大容量液体灌洗协同作用的优势,是治疗胃癌PC的有效方法。多项国内外临床研究结果表明,CRS+HIPEC可延缓PC患者肿瘤复发、提高远期生活质量[7-111。尽管CRS+HIPEC治疗PC已在临床应用多年,显示出临床治疗优势,但HIPEC相关的基础研究却有待深化,高水平的实验室研究结果很少,缺少足够的循证医学证据[12-14]。构建合理可靠的动物模型是进行疗效学研究的重要平台。近年来国内外相继构建了裸小鼠PC模型、小鼠PC模型和大鼠PC模型,用于PC的疗效学等研究。但由于动物体积小、循环血量少等原因,裸小鼠PC模型和小鼠PC模型仅适于非手术治疗研究,不能用于CRS、CRS+HIPEC等包含手术治疗措施的治疗策略的疗效评价,而使应用受限。大鼠动物体积相对较大,循环血量相对较多,对手术有一定的耐受能力,但仍然难以承受更大范围的手术及其他治疗。目前国内外尚无构建兔胃癌PC动物模型的报道,亦无CRS加术中HIPEC治疗胃癌PC的动物实验报道。在胃癌进展过程中,癌细胞可合成分泌组织蛋白酶B(Cathepsin B, Cat B),破坏细胞外基质,并促进癌细胞在腹膜表面种植和形成克隆,进而形成胃癌PC。Cat B可作为治疗胃癌PC的重要候选靶点。阿霉素(Doxorubicin,DOX)是重要的蒽环类抗生素,对胃癌等恶性肿瘤疗效显著,但也因其心脏毒性、骨髓抑制等毒副作用显著而使应用受限。本课题组利用胃癌等恶性肿瘤侵袭转移过程中局部释放Cat B的生物学特点,设计了新型阿霉素前体药(Peptide Doxorubicin, PDOX)[18-2,通过空间构象分子PABC在DOX分子上连接Cat B的特异底物Ac-Phe-Lys,以实现减毒增效。在正常组织和外周血中Cat B存在于细胞的溶酶体中,PDOX无活性,但在恶性肿瘤侵袭转移过程中,癌细胞分泌大量Cat B[21-22],降解Ac-Phe-Lys,随后PABC自水解[23],释放出游离DOX分子,杀灭周围癌细胞[18]。本研究内容分为三部分：第一部分：兔VX2腹膜转移癌模型的建立及转移特征研究目的：建立稳定的大动物(兔)胃癌PC模型,并鉴定其生物学行为,为开展大型手术在内的PC综合治疗策略等研究提供可靠的动物实验平台。方法：将36只新西兰大白兔随机分为三组,取VX2瘤组织制备1mm3瘤组织块和VX2瘤细胞悬液后,分别以下述三种方法建立兔胃癌腹膜癌模型：A组,以开腹穿刺种植法种瘤,开腹后将VX2瘤细胞悬液经胃窦部浆膜穿刺注入胃窦部粘膜下(n=12)；B组,以开腹包埋种植法种瘤,开腹后将肿瘤组织块包埋种植于兔胃窦部大网膜内(n=12)；C组,以直接经皮穿刺种植法种瘤,不行开腹手术,直接腹腔穿刺注入VX2瘤细胞悬液(n=12)。造模后,每天观察各组动物肿瘤生长状况,并通过病理学和影像学检查分析各组动物局部区域及远处转移情况。结果：三种方法构建模型的成功率分别是100%(12/12)、91.7%(11/12)和58.3%(7/12),与经皮穿刺法比较,开腹包埋法造模成功率明显提高(P<0.05)；与开腹包埋法比较,开腹穿刺法造模成功率进一步明显提高(P<0.05)。成功构建的兔胃癌腹膜癌模型病理发展进程与人胃癌腹膜癌病理过程相似,接种1～2周后,形成典型溃疡性胃癌并区域性腹膜转移癌表现,精神、饮食、活动逐渐变差,4周时动物逐渐衰竭死亡,部分动物出现肺转移,病理学检查显示图胃癌腹膜癌病理特点符合典型的人胃癌腹膜癌病理学特点。结论：开腹穿刺法制作兔VX2胃癌腹膜转移癌模型成功率最高,制作过程简单,实验周期短,其病理发展进程与人胃癌腹膜癌病理过程相似,临床病理表现类似人类腹膜转移癌,为开展胃癌腹膜癌的实验研究提供可靠的大动物模型。第二部分：新型阿霉素前体药PDOX联合CRS+HIPEC治疗兔胃癌PC的疗效及安全性研究目的：在成功建立大动物(兔)胃癌PC模型基础上,研究CRS+HIPEC治疗兔胃癌PC的疗效和安全性,为CRS+HIPEC治疗胃癌PC的临床研究和应用提供理论支持和循证医学依据。方法：以成年雄性新西兰兔为研究对象,以开腹穿刺种植法将VX2癌细胞注射入胃窦部粘膜下层,形成兔胃癌PC模型,42只新西兰大白兔随机分为空白对照组(n=14),单纯CRS组(n=14),CRS+HIPEC组(n=14)。在接种肿瘤后第8～9d进行干预治疗,HIPEC药物为多西紫杉醇(10mg/只)、卡铂(40mg/只),加热至42℃,行腹腔灌注30分钟。主要疗效指标为生存期,次要疗效指标为体重、生化指标及安全性。结果：模型制作成功率为100%(42/42),动物生存期在空白对照组为24d(8～30d)；单纯CRS组为27d(20～40d)；CRS+HIPEC组为46d(23～55d)(单纯CRS组vs.空白对照组P=0.1133；CRS+HIPEC组vs.空白对照组P=2.45×10-6;CRS+HIPEC组VS.单纯CRS组P=0.0012)。与单纯CRS相比,HIPEC至少能使生存期延长70%。种瘤后三组动物均出现体重下降,与空白对照组及CRS组相比,HIPEC组动物体重变化特点是：治疗后早期体重明显下降,3～5d后体重下降减缓,约10d后体重再次出现下降趋势,提示HIPEC可延缓肿瘤所致的体重减轻。在肿瘤接种前、手术前及手术后第7d,各组的外周血、肝肾功能及血生化指标均无显著性差异。严重不良事件在空白对照组为0只；CRS组为2只,其中1只为麻醉意外死亡,另1只术后第2d腹腔大出血死亡；CRS+HIPEC组为3只,1只为麻醉意外死亡,另2只在分别术后23、27d因急性腹泻死亡。结论：对胃癌PC大动物模型,单纯CRS并不能改善预后,而CRS+HIPEC则能显著延长生存期,安全性可以接受。第三部分新型阿霉素前体药PDOX对胃癌腹膜转移癌模型的分子靶向治疗研究目的：合成新型阿霉素前体药PDOX,并利用成功构建的大动物(兔)胃癌PC模型,研究新型阿霉素前体药PDOX联合CRS+HIPEC治疗兔胃癌PC的疗效和安全性,评估PDOX靶向治疗胃癌PC的潜力并探讨可能存在的毒副反应。方法：通过7个化学步骤合成PDOX后,取成年雄性新西兰大白兔40只,将VX2瘤细胞悬液(约5×106个瘤细胞/0.1mL/兔)注入胃窦部粘膜下层,制成溃疡型胃癌PC模型,随机分为4组：Control组(n=10)不行任何治疗；HIPEC组(n=10)种瘤后第8d行CRS+HIPEC(多西紫杉醇10.0mg+卡铂50.0mg,42℃持续腹腔灌注30min)；PDOX组(n=10)种瘤后第8d行CRS+HIPEC,第16d开始PDOX静脉化疗,每4d一次,共五次,每次给予PDOX10.0mg/kg,总剂量为50.0mg/kg；DOX组(n=10)种瘤后第8d行CRS+HIPEC,第16d开始DOX静脉化疗,每4d一次,共五次,每次给予DOX1.0mg/kg,总剂量为5.0mg/kg。定期观察动物一般状况,称体质量和检测血液指标。动物自然死亡后解剖记录胃肿瘤特点、腹腔内转移特点、全身转移特点、重要脏器病变情况等。本研究主要疗效指标为生存期,次要疗效指标为荷瘤程度和安全性。结果：合成的PDOX为红色固体,纯度99.1%,易溶于水,稳定性好。各组动物的中位生存期在Control组为23.0d(95%CI:19.9～26.1d),HIPEC组41.0d(36.9～45.1d),PDOX组65.0d(44.1～71.9d),DOX组58.0d(39.6～54.4d)。HIPEC组生存期较Control组延长70%(18d)以上(P<0.001),PDOX组较HIPEC组延长58%(24d)(P=0.021),DOX组较HIPEC组延长40%(17d)(P=0.029)。DOX组化疗后(D36)WBC、PLT明显低于HIPEC组(P=0.001),CK-MB明显高于HIPEC组(P=0.012),其余各组间血液学指标无统计学差异(P>0.05)。与Control组比较,HIPEC组PC程度减轻,肺转移概率增高；PDOX组和DOX组PC程度进一步降低,且肺转移概率无明显增高。DOX组光镜下见心肌凝固性坏死并核固缩,心肌组织透射电子显微照片(Transimission electronic micrograph, TEM)显示提示心肌细胞核退行性变,核周线粒体减少或消失,肌丝部分溶解等,其余组则未见上述改变。结论：新型阿霉素前体药PDOX纯度高、理化性质优良。在CRS+HIPEC基础上,联合PDOX可进一步延长荷瘤动物生存期,安全性好。与DOX比,PDOX具有高效低毒的特点。PDOX具有进一步开发的价值。更多还原

【Abstract】 The loco-regional progression of gastric cancer usually results in peritoneal carcinomatosis (PC), characterized by the presence of tumor nodules of various size, number, and distribution on the peritoneal surface as well as malignant ascites, with very poor prognosis and a median survival of less than6months.The most widely accepted therapies for such PC are surgery alone plus chemotherapy. Surgery alone, however, can only remove the bulky visible tumor nodules. For the micrometastases, invisible free cancer cells and those tumor masses not suitable for resection, surgery can not achieve any effect. Therefore, the traditional therapy can not make obvious positive impact on the survival and quality of life in patients with PC.In order to tackle this difficult problem, a new treatment strategy called cytoreductive surgery (CRS) plus hyperthermic intraperitoneal chemotherapy (HIPEC) has been developed over the past three decades, which has the advantages of surgery to reduce the visible tumor burden and regional hyperthermic chemotherapy to eradicate micrometastses and free cancer cells.Although many clinical studies have been performed to test and confirm the efficacy of this combined treatment approach, there is a lack of high quality evidence from phase Ⅲ randomized prospective studies. In order to more objectively evaluate such treatment, it is necessary to study this treatment modality under experimental conditions, in which most of the confounding factors could be well controlled. In this respect, suitable animal models of PC are indispensable platforms. Small animal models of PC have been established, including nude mouse PC models, mouse PC models and rat PC models. In most of these animal models, cancer cells are injected directly into the peritoneum, which will result in widespread PC in due time. Such models have been used to test various treatment modalities, including CRS and HIPEC, either alone or in combination, producing valuable information on the validity of different therapies. The small body size and delicate hemodynamic conditions are limiting factors for complex surgical interventions. Large animal models of PC might be more suitable for extensive surgical treatment. Therefore, it is necessary to establish large animal model of gastric cancer with PC for experimental studies to test extensive CRS and HIPEC.During the development of peritoneal carcinomatosis, GC cells secret enzymes to facilitate cancer cells seeding and colonization on the peritoneum. Cathepsin B is among the key enzymes in this critical process, overexpressed in GC as well as other cancers,10-12and actively involved in cancer invasion. Conversely, its expression is extremely low in normal cells, and it is inactive or loses activity as soon as it is dispersed in aqueous media away from cells. Thus, cathepsin B has long been considered a candidate target in cancer therapy [16].Doxorubicin (DOX) is a typical representative of anthracyclines. Although doxorubicin is an important drug in chemotherapy, its toxicities are also well known, such as cardiac toxicities and bone marrow suppression [17]. To retain the therapeutic effect while reducing the side effects, we designed a smart prodrug of doxorubicin, PDOX (Ac-Phe-Lys-PABC-DOX)[18-20]. In this modified doxorubicin, Phe-Lys is a dipeptide specific for cathepsin B, and PABC (para-aminobenzyloxycarbonyl) is a self-immolative spacer [21]. The prodrug is inactive when there is little cathepsin B activity, such as in normal tissues and peripheral blood, thus avoiding the side effects on normal tissue. During cancer invasion, activated cathepsin B is overexpressed on the exterior membrane of the invading cancer cells, which cleaves the Phe-Lys dipeptide at the Lys-PABC bond [22]. Then the exposed PABC spacer can self-hydrolyze upon deacylation[23], and free doxorubicin molecules are released, resulting in direct killing of the invading cancer cells[16].This study was divided into three parts as below:Part One:Establishment and Identification of Rabbit Model of Gastric Cancer with Peritoneal CarcinomatosisObjective To establish a stable model of peritoneal carcinomatosis in rabbit using VX2tumor and analyzed the features of metastasis.Method VX2tumor was implanted into36New Zealand rabbits by3methods:laparotomic orthotopic injection of cancer cells into the submucosal layer of the stomach (Group A), laparotomic implantation of tumor tissue into the greater omentum immediately beneath the gastric antrum (Group B), and percutaneous injetion of tumor cells directly into the peritoneal cavity (Group C),12rabbits were randamized aranged in each group. The animals wereclosely observed and detailed clinico-pathological studies were conducted. Result The success rates of peritoneal carcinomatosis formation were100%(12/12),91.7%(11/12) and58.3%(7/12), respectively, for Groups A, B and C (P=0.019, A vs. C; P=0.077, B vs. C; P=0.500, A vs. B, Fisher’s exact test). Two weeks after submucosal cancer cells injection in Group A, ulcerative gastric cancer with peritoneal carcinomatosis showed typical VX2tumor pathology, with widespread intraperitoneal metastatic nodules, bloody asites and perspicuous pulmonary metastases. The clinicalopathological progression pattern was very similar to patients of advanced gastric cancer with peritoneal carcinomatosis.Conclusion The orthotopic tissue implantation to establish a peritoneal carcinomatosis modelis convenient, feasible and timesaving. The clinicalpathological features of the model are similar to those of peritoneal carcinomatosis. Part Two:Experimental Animal Study on The Efficacy and Safty of Cytoreductive Surgery plus Hyperthermic Intraperitoneal Chemotherapy to Treat Peritoneal Carcinomatosis from Gastric CancerObjective To study the efficacy and safety of cytoreductive surgery (CRS) with hyperthermic introperitoneal chemotherapy (HIPEC) to treat rabbit model of gastric cancer with peritoneal carcinomatosis (PC), so as to provide support to clinical application.Method VX2tumor cells were injected into the gastric submucosa of42adult male New Zealand rabbits using a laparotomic implantation technique, to construct rabbit model of gastric cancer with PC. The rabbits were randomly divided into three groups:Control group (n=14), CRS group (n=14), and CRS+HIPEC group (n=14). The Control group was observed for natural course of disease progression. Treatment was initiated8ro9days after tumor cells inoculation, including optimal removal of tumor nodules in CRS group, and maximal removal of tumor nodules and heperthermic chemoperfusion in the CRS+HIPEC group with docetaxel (10mg/rabbit) and carboplatin (40mg/rabbit) at42℃for30minutes. The primary endpoint was overall survival in each group. The secondary endpoints were body weight, biochemistry, major organ functions and serious adverse events.Result The success rates of rabbit PC model were100%(42/42). The clinicopathological feature of the model is similar to peritoneal carcinomatosis in human. Overall survival was18-30days (median24days) in Control group,20~40days (median27days) in CRS group; and23~55days (median46days) in CRS plus HIPEC group (CRS alone group vs. control group P=0.1133; CRS+HIPEC group vs. control group P=2.45x10-6; CRS+HIPEC group vs. pure CRS group P=0.0012). Compared with CRS only or Control group, HIPEC could extend the overall survival by at least60%. At the baseline, on the day of surgery and7days after surgery, the peripheral blood cells counts, liver and renal functions, and biochemistry parameters were all comparable. Serious adverse events occurred in0animal in Control group, 2animals in CRS alone group including1animal died of anesthesia overdose and another1died of postoperative hemorrhage, and3animlas in CRS+HIPEC group including1animal died of anesthesia overdose, and2died of diarrhea23and27days after operation.Conclusion For rabbit model of gastric cancer PC, CRS alone could not bring benefit while CRS+HIPEC with docetaxel and cisplatin could significantly prolong the survival with a acceptable safety. Part III:Synthesis, Identification and In Vivo Studies of Tumor-Targeting Agent Peptide Doxorubicin (PDOX) to Treat Peritoneal Carcinomatosis of Gastric Cancer with Similar Efficacy and Reduced ToxicityObjective This work aimed to synthesize a cathepsin B (Cat B)-cleavable tumor-targeting prodrug peptide doxorubicin (PDOX) and study the in vivo efficacy and toxicities on an animal model of gastric cancer (GC) with peritoneal carcinomatosis (PC).Methods PDOX was synthesized using doxorubicin (DOX) attaching to a Cat B-cleavable dipeptide Ac-Phe-Lys and a para-amino-benzyloxycarbonyl (PABC) spacer. PC model was established by injecting VX2tumor cells into the gastric sub-mucosa of40rabbits, which then were randomized into4groups:the Control (n=10) without treatment, the HIPEC (n=10) receiving cytoreductive surgery (CRS) plus hyperthermic intraperitoneal chemotherapy (HIPEC), the PDOX (n=10) and the DOX (n=10) receiving systemic chemotherapy with PDOX50.0mg/kg or DOX5.0mg/kg after CRS+HIPEC respectively.Results The median overall survivals (OS) were23.0d (95%CI:19.9d-26.1d) in the Control,41.0d (36.9d-45.1d) in the HIPEC,65.0d (44.1d-71.9d) in the PDOX, and58.0d (39.6d-54.4d) in the DOX. Compared with the Control, the OS was extended by70%in the HIPEC (P<0.001) and further extended by40%in the DOX (P=0.029) and by58%in the PDOX (P=0.021), the PC severity was decreased in the HIPEC and further decreased in the PDOX and DOX. Animals receiving DOX treatment showed hematological toxicities with marked reduction of white blood cells and platelets, as well as cardiac toxicities with significant increases in creatine kinase mb isoenzyme, evident myocardium coagulation necrosis, significant nuclear degeneration, peri-nucleus mitochondria deletion, mitochondria-pyknosis, and abnormal intercalated discs. But these toxicities were not evident in the PDOX.Conclusions PDOX is a newly synthesized tumor-targeting prodrug of DOX. Compared with DOX, PDOX has similar efficacy and reduced hematological and cardiac toxicities in treating rabbit model of GC with PC.更多还原