新型阿霉素前体药PDOX联合细胞减灭术加腹腔热灌注化疗治疗胃癌腹膜转移癌的实验研究

发布时间：2018-01-15 17:06

  本文关键词：新型阿霉素前体药PDOX联合细胞减灭术加腹腔热灌注化疗治疗胃癌腹膜转移癌的实验研究　出处：《武汉大学》2014年博士论文　论文类型：学位论文

  更多相关文章： 胃癌 腹膜转移癌 动物模型 兔胃癌PC 胃癌 腹膜转移癌 CRS HIPEC 兔胃癌PC 胃癌 腹膜转移癌 分子靶向治疗 PDOX CRS+HIPEC

【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(P0.05)；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(P0.05)。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(P0.001),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(P0.05)。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。
[Abstract]:Gastric cancer, malignant tumor prone to local progress, often lead to peritoneal metastatic carcinoma (peritonealcarcinomatosis, PC). PC gastric cancer prognosis, with a median survival of approximately 6 months. The traditional treatment for gastric cancer PC in radical resection of gastric cancer, comprehensive treatment combined with systemic chemotherapy. However, visible to the naked eye tumor resection surgery alone, unable to clear intraperitoneal residual cancer cells [4-6]. therefore, not traditional treatment method to prolong the survival of patients with PC.
Cytoreductive surgery (cytoreductive surgery, CRS) intraperitoneal hyperthermic perfusion chemotherapy plus surgery (hyperthermicintraperitoneal chemotherapy, HIPEC) to explore in the world for nearly 30 years, first through the CRS visible tumor resection, and then through the clearance of intraperitoneal residual tumor and micro free cancer cells in HIPEC patients, in radical surgery, regional chemotherapy, hyperthermia and large capacity liquid filling synergy advantages, is an effective method for the treatment of gastric cancer PC. A number of domestic and international clinical research results show that CRS+HIPEC can delay tumor recurrence of PC patients, improve the long-term quality of life [7-111.
Although CRS+HIPEC treatment of PC has been used for many years and showed clinical advantages, but the basic research related to HIPEC is to be deepened, laboratory research results of high level small animal model of the lack of reasonable and reliable curative effect is an important platform for the study of evidence-based medicine [12-14]. enough. In recent years at home and abroad have been constructed the nude mice model of PC mice, PC model and PC model rats, to study the effect of PC and so on. But because the animal has the advantages of small volume, less blood circulation, nude mice model of PC mice and PC model is only suitable for the treatment of non operation, cannot be used for CRS, CRS+HIPEC and clinical evaluation of treatment strategies including surgical treatment the measures, the application is limited. The rat animal relatively large volume of circulating blood volume is relatively large, there is a certain tolerance of surgery, but it is still difficult to bear a greater range of surgery and other treatment There is no report on the construction of PC animal model of rabbit gastric cancer at home and abroad, and there is no animal experiment report on the treatment of gastric cancer PC by HIPEC in the operation of CRS.
In the progress of gastric cancer, cancer cells can synthesis and secretion of cathepsin B (Cathepsin B, Cat B), disruption of the extracellular matrix, and promote the cultivation of cancer cells and cloning formation in the peritoneal surface, and the formation of gastric cancer PC.Cat B can be used as an important candidate target for the treatment of gastric cancer PC. Adriamycin (Doxorubicin, DOX) is an important anthracyclines, significant effect on gastric cancer and other malignant tumors, but also because of the cardiac toxicity, side effects such as bone marrow suppression was due to the limited.
The research group with gastric cancer and so on biological characteristics of local release of Cat B invasion, designed a new type of adriamycin prodrug (Peptide Doxorubicin, PDOX [18-2), Ac-Phe-Lys Cat B specific substrate connection on DOX molecules through the conformation of PABC molecules, in order to achieve synergistic in normal tissue and peripheral. The blood Cat B exists in the cell lysosomes, PDOX activity, but in the process of metastasis of malignant tumor invasion, tumor cells secrete Cat B[21-22], degradation of Ac-Phe-Lys, then PABC from the hydrolysis of [23], a free DOX molecule, killing cancer cells around [18].
The content of this study is divided into three parts:
Part one: Study on the establishment and metastasis of VX2 peritoneal metastasis model in rabbits
Objective: to establish a stable large animal (rabbit) gastric cancer PC model and identify its biological behavior, so as to provide reliable animal experimental platform for PC comprehensive treatment strategy, including large-scale operation.
Methods: 36 New Zealand rabbits were randomly divided into three groups, VX2 tumor tissue preparation 1mm3 tumor tissue and VX2 tumor cell suspension, respectively, the following three methods to establish the rabbit model of gastric cancer peritoneal cancer: A group, to open the puncture method of planting tumor, open after VX2 tumor cell suspension injected into the gastric antrum after gastric antrum puncture under serosa (n=12); group B, to open the embedding method of planting tumor, open after tumor tissue embedding cultivated in rabbit gastric antrum in the greater omentum (n=12); group C, with direct percutaneous implantation of tumor, not open abdominal cavity puncture surgery, direct injection of VX2 tumor cell suspension (n=12). After modeling, every observed animal tumor growth situation, and through the pathology and imaging analysis of each animal local and distant metastasis.
Results: the three methods of constructing model success rate were 100% (12/12), 91.7% (11/12) and 58.3% (7/12), and percutaneous puncture method, open embedding model significantly improved the success rate (P0.05); and open embedding method, abdominal puncture success rate of the model into a step increased significantly (P0.05). Rabbit peritoneal cancer pathological model development and human gastric cancer peritoneal cancer pathological process successfully constructed similar, 1~2 weeks after inoculation, the typical form of ulcerative gastric cancer and regional peritoneal metastatic carcinoma, spirit, diet, activity gradually becomes poor, 4 weeks of animal gradually failure death, part of animal lung metastasis, pathological examination showed that the pathological characteristics of gastric cancer with peritoneal cancer figure typical human gastric cancer peritoneal carcinomatosis
The characteristics of science.
Conclusion: abdominal puncture of rabbits were VX2 gastric cancer peritoneal metastasis model of the highest success rate, simple production process, short experimental period, the pathological process and human gastric cancer peritoneal cancer pathological process similar to clinical and pathological manifestations of human peritoneal metastatic carcinoma, provide a reliable animal model for experimental study of gastric cancer peritoneal cancer. The second part: To study the efficacy and safety of the novel doxorubicin prodrug of PDOX combined with CRS+HIPEC in the treatment of rabbit gastric cancer PC
Objective: Based on the successful establishment of a large animal (rabbit) gastric cancer PC model, we studied the efficacy and safety of CRS+HIPEC in the treatment of gastric cancer PC in rabbits, and provided theoretical support and evidence-based medicine for the clinical research and application of CRS+HIPEC in the treatment of gastric cancer PC.
Methods: adult male New Zealand rabbits as the research object, to open the VX2 puncture planting method of cancer cells injected into the submucosa of gastric antrum, gastric cancer formation in rabbit PC model, 42 New Zealand white rabbits were randomly divided into control group (n=14), CRS group (n=14), CRS+HIPEC group (n=14) to eighth. At 9D after tumor inoculation treatment, HIPEC drug docetaxel (10mg/), carboplatin (40mg/), heated to 42 DEG C for 30 minutes. The main effect of intraperitoneal perfusion index for survival, the secondary index weight, biochemical index and safety.
Results: the model success rate is 100% (42/42), animal survival in blank control group was 24D (8 ~ 30d); CRS group was 27D (20 ~ 40d); CRS+HIPEC group was 46d (23 ~ 55D) (pure CRS group vs. control group P=0.1133; group CRS+HIPEC vs. control group P=2.45 * 10-6; group CRS+HIPEC VS. CRS group P=0.0012). Compared with CRS, HIPEC can prolong the survival of at least 70%. tumor after three animal groups showed decreased body weight, compared with the blank control group and CRS group, HIPEC group is the characteristics of weight change: after treatment of early animal body weight was significantly decreased after 3 ~ 5D weight loss decreased, about 10d weight again decreased, suggesting that HIPEC can delay tumor induced weight loss. In the tumor before inoculation, preoperative and postoperative 7d, peripheral blood of each group, there were no significant differences in liver and kidney function and blood biochemical indexes. Serious adverse events in the control Group 0, group CRS, 2, 1 of them were anesthetized, and 1 died of abdominal bleeding after 2D, 3 in group CRS+HIPEC, 1 in anesthetic accident, and 2 in 23,27d after operation.
Conclusion: for the large PC animal model of gastric cancer, simple CRS can not improve the prognosis, while CRS+HIPEC can prolong the survival time significantly, and the safety is acceptable.
The molecular targeting therapy of the third new adriamycin prodrug PDOX for gastric carcinoma model of peritoneal metastasis
Objective: to synthesize novel doxorubicin prodrug of PDOX, and the successful construction of large animal (rabbit) gastric cancer PC model, study the efficacy and safety of the new adriamycin prodrug of PDOX combined with CRS+HIPEC in the treatment of rabbit gastric cancer PC, PDOX evaluation of targeted therapy of gastric carcinoma PC and to explore the potential side effects may exist.
Methods: through 7 steps of the synthesis of chemical PDOX, adult male and 40 New Zealand rabbits, VX2 tumor cell suspension (5 * 106 cells /0.1mL/ rabbit) injection in gastric antrum submucosa, made of ulcerated gastric cancer PC model, were randomly divided into 4 groups: group Control (n=10) without any treatment; group HIPEC (n=10) tumor after 8D CRS+HIPEC (10.0mg+ 50.0mg 42 C docetaxel carboplatin, continuous hyperthermic peritoneal perfusion 30min); group PDOX (n=10) tumor after 8D CRS+HIPEC, the 16d began to PDOX chemotherapy, every 4D, a total of five times, each time for PDOX10.0mg/kg, the total dose of 50.0mg/kg; group DOX (n=10) tumor after 8D CRS+HIPEC, the 16d began to DOX chemotherapy, every 4D, a total of five times, each time for DOX1.0mg/kg, the total dose of 5.0mg/kg. regularly to observe the general situation of animal, called the body mass and blood indices. The anatomical record of gastric tumor characteristics of natural animal after death, The characteristics of intraperitoneal metastasis, the characteristics of systemic metastasis and the condition of important organs are the main indicators of survival. The secondary efficacy is the degree and safety of tumor bearing.
缁撴灉锛氬悎鎴愮殑PDOX涓虹孩鑹插浐浣，

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