本文關鍵詞：新型阿霉素前體藥PDOX聯(lián)合細胞減滅術加腹腔熱灌注化療治療胃癌腹膜轉移癌的實驗研究　出處：《武漢大學》2014年博士論文　論文類型：學位論文

  更多相關文章： 胃癌 腹膜轉移癌 動物模型 兔胃癌PC 胃癌 腹膜轉移癌 CRS HIPEC 兔胃癌PC 胃癌 腹膜轉移癌 分子靶向治療 PDOX CRS+HIPEC

【摘要】：胃癌在內的腹盆腔惡性腫瘤易發(fā)生局域性進展,往往導致腹膜轉移癌(peritonealcarcinomatosis, PC)。胃癌PC預后極差,其中位生存期約6個月。針對胃癌PC的傳統(tǒng)治療手段是以胃癌根治術為主,輔以全身化療的綜合治療。然而,單純手術僅切除肉眼可見腫瘤組織,無法清除腹腔內殘余癌細胞[4-6]。因此,傳統(tǒng)治療方法不能有效延長PC患者的生存期。 細胞減滅術(cytoreductive surgery, CRS)加術中腹腔熱灌注化療(hyperthermicintraperitoneal chemotherapy,HIPEC)在國際上探索了近30年,首先通過CRS切除肉眼可見瘤灶,再通過術中HIPEC清除腹腔內殘余微癌灶和游離癌細胞,集根治手術、區(qū)域性化療、熱療和大容量液體灌洗協(xié)同作用的優(yōu)勢,是治療胃癌PC的有效方法。多項國內外臨床研究結果表明,CRS+HIPEC可延緩PC患者腫瘤復發(fā)、提高遠期生活質量[7-111。 盡管CRS+HIPEC治療PC已在臨床應用多年,顯示出臨床治療優(yōu)勢,但HIPEC相關的基礎研究卻有待深化,高水平的實驗室研究結果很少,缺少足夠的循證醫(yī)學證據(jù)[12-14]。構建合理可靠的動物模型是進行療效學研究的重要平臺。近年來國內外相繼構建了裸小鼠PC模型、小鼠PC模型和大鼠PC模型,用于PC的療效學等研究。但由于動物體積小、循環(huán)血量少等原因,裸小鼠PC模型和小鼠PC模型僅適于非手術治療研究,不能用于CRS、CRS+HIPEC等包含手術治療措施的治療策略的療效評價,而使應用受限。大鼠動物體積相對較大,循環(huán)血量相對較多,對手術有一定的耐受能力,但仍然難以承受更大范圍的手術及其他治療。目前國內外尚無構建兔胃癌PC動物模型的報道,亦無CRS加術中HIPEC治療胃癌PC的動物實驗報道。 在胃癌進展過程中,癌細胞可合成分泌組織蛋白酶B(Cathepsin B, Cat B),破壞細胞外基質,并促進癌細胞在腹膜表面種植和形成克隆,進而形成胃癌PC。Cat B可作為治療胃癌PC的重要候選靶點。阿霉素(Doxorubicin,DOX)是重要的蒽環(huán)類抗生素,對胃癌等惡性腫瘤療效顯著,但也因其心臟毒性、骨髓抑制等毒副作用顯著而使應用受限。 本課題組利用胃癌等惡性腫瘤侵襲轉移過程中局部釋放Cat B的生物學特點,設計了新型阿霉素前體藥(Peptide Doxorubicin, PDOX)[18-2,通過空間構象分子PABC在DOX分子上連接Cat B的特異底物Ac-Phe-Lys,以實現(xiàn)減毒增效。在正常組織和外周血中Cat B存在于細胞的溶酶體中,PDOX無活性,但在惡性腫瘤侵襲轉移過程中,癌細胞分泌大量Cat B[21-22],降解Ac-Phe-Lys,隨后PABC自水解[23],釋放出游離DOX分子,殺滅周圍癌細胞[18]。 本研究內容分為三部分： 第一部分：兔VX2腹膜轉移癌模型的建立及轉移特征研究 目的：建立穩(wěn)定的大動物(兔)胃癌PC模型,并鑒定其生物學行為,為開展大型手術在內的PC綜合治療策略等研究提供可靠的動物實驗平臺。 方法：將36只新西蘭大白兔隨機分為三組,取VX2瘤組織制備1mm3瘤組織塊和VX2瘤細胞懸液后,分別以下述三種方法建立兔胃癌腹膜癌模型：A組,以開腹穿刺種植法種瘤,開腹后將VX2瘤細胞懸液經(jīng)胃竇部漿膜穿刺注入胃竇部粘膜下(n=12)；B組,以開腹包埋種植法種瘤,開腹后將腫瘤組織塊包埋種植于兔胃竇部大網(wǎng)膜內(n=12)；C組,以直接經(jīng)皮穿刺種植法種瘤,不行開腹手術,直接腹腔穿刺注入VX2瘤細胞懸液(n=12)。造模后,每天觀察各組動物腫瘤生長狀況,并通過病理學和影像學檢查分析各組動物局部區(qū)域及遠處轉移情況。 結果：三種方法構建模型的成功率分別是100%(12/12)、91.7%(11/12)和58.3%(7/12),與經(jīng)皮穿刺法比較,開腹包埋法造模成功率明顯提高(P0.05)；與開腹包埋法比較,開腹穿刺法造模成功率進一步明顯提高(P0.05)。成功構建的兔胃癌腹膜癌模型病理發(fā)展進程與人胃癌腹膜癌病理過程相似,接種1～2周后,形成典型潰瘍性胃癌并區(qū)域性腹膜轉移癌表現(xiàn),精神、飲食、活動逐漸變差,4周時動物逐漸衰竭死亡,部分動物出現(xiàn)肺轉移,病理學檢查顯示圖胃癌腹膜癌病理特點符合典型的人胃癌腹膜癌病 理學特點。 結論：開腹穿刺法制作兔VX2胃癌腹膜轉移癌模型成功率最高,制作過程簡單,實驗周期短,其病理發(fā)展進程與人胃癌腹膜癌病理過程相似,臨床病理表現(xiàn)類似人類腹膜轉移癌,為開展胃癌腹膜癌的實驗研究提供可靠的大動物模型。第二部分：新型阿霉素前體藥PDOX聯(lián)合CRS+HIPEC治療兔胃癌PC的療效及安全性研究 目的：在成功建立大動物(兔)胃癌PC模型基礎上,研究CRS+HIPEC治療兔胃癌PC的療效和安全性,為CRS+HIPEC治療胃癌PC的臨床研究和應用提供理論支持和循證醫(yī)學依據(jù)。 方法：以成年雄性新西蘭兔為研究對象,以開腹穿刺種植法將VX2癌細胞注射入胃竇部粘膜下層,形成兔胃癌PC模型,42只新西蘭大白兔隨機分為空白對照組(n=14),單純CRS組(n=14),CRS+HIPEC組(n=14)。在接種腫瘤后第8～9d進行干預治療,HIPEC藥物為多西紫杉醇(10mg/只)、卡鉑(40mg/只),加熱至42℃,行腹腔灌注30分鐘。主要療效指標為生存期,次要療效指標為體重、生化指標及安全性。 結果：模型制作成功率為100%(42/42),動物生存期在空白對照組為24d(8～30d)；單純CRS組為27d(20～40d)；CRS+HIPEC組為46d(23～55d)(單純CRS組vs.空白對照組P=0.1133；CRS+HIPEC組vs.空白對照組P=2.45×10-6;CRS+HIPEC組VS.單純CRS組P=0.0012)。與單純CRS相比,HIPEC至少能使生存期延長70%。種瘤后三組動物均出現(xiàn)體重下降,與空白對照組及CRS組相比,HIPEC組動物體重變化特點是：治療后早期體重明顯下降,3～5d后體重下降減緩,約10d后體重再次出現(xiàn)下降趨勢,提示HIPEC可延緩腫瘤所致的體重減輕。在腫瘤接種前、手術前及手術后第7d,各組的外周血、肝腎功能及血生化指標均無顯著性差異。嚴重不良事件在空白對照組為0只；CRS組為2只,其中1只為麻醉意外死亡,另1只術后第2d腹腔大出血死亡；CRS+HIPEC組為3只,1只為麻醉意外死亡,另2只在分別術后23、27d因急性腹瀉死亡。 結論：對胃癌PC大動物模型,單純CRS并不能改善預后,而CRS+HIPEC則能顯著延長生存期,安全性可以接受。 第三部分新型阿霉素前體藥PDOX對胃癌腹膜轉移癌模型的分子靶向治療研究 目的：合成新型阿霉素前體藥PDOX,并利用成功構建的大動物(兔)胃癌PC模型,研究新型阿霉素前體藥PDOX聯(lián)合CRS+HIPEC治療兔胃癌PC的療效和安全性,評估PDOX靶向治療胃癌PC的潛力并探討可能存在的毒副反應。 方法：通過7個化學步驟合成PDOX后,取成年雄性新西蘭大白兔40只,將VX2瘤細胞懸液(約5×106個瘤細胞/0.1mL/兔)注入胃竇部粘膜下層,制成潰瘍型胃癌PC模型,隨機分為4組：Control組(n=10)不行任何治療；HIPEC組(n=10)種瘤后第8d行CRS+HIPEC(多西紫杉醇10.0mg+卡鉑50.0mg,42℃持續(xù)腹腔灌注30min)；PDOX組(n=10)種瘤后第8d行CRS+HIPEC,第16d開始PDOX靜脈化療,每4d一次,共五次,每次給予PDOX10.0mg/kg,總劑量為50.0mg/kg；DOX組(n=10)種瘤后第8d行CRS+HIPEC,第16d開始DOX靜脈化療,每4d一次,共五次,每次給予DOX1.0mg/kg,總劑量為5.0mg/kg。定期觀察動物一般狀況,稱體質量和檢測血液指標。動物自然死亡后解剖記錄胃腫瘤特點、腹腔內轉移特點、全身轉移特點、重要臟器病變情況等。本研究主要療效指標為生存期,次要療效指標為荷瘤程度和安全性。 結果：合成的PDOX為紅色固體,純度99.1%,易溶于水,穩(wěn)定性好。各組動物的中位生存期在Control組為23.0d(95%CI:19.9～26.1d),HIPEC組41.0d(36.9～45.1d),PDOX組65.0d(44.1～71.9d),DOX組58.0d(39.6～54.4d)。HIPEC組生存期較Control組延長70%(18d)以上(P0.001),PDOX組較HIPEC組延長58%(24d)(P=0.021),DOX組較HIPEC組延長40%(17d)(P=0.029)。DOX組化療后(D36)WBC、PLT明顯低于HIPEC組(P=0.001),CK-MB明顯高于HIPEC組(P=0.012),其余各組間血液學指標無統(tǒng)計學差異(P0.05)。與Control組比較,HIPEC組PC程度減輕,肺轉移概率增高；PDOX組和DOX組PC程度進一步降低,且肺轉移概率無明顯增高。DOX組光鏡下見心肌凝固性壞死并核固縮,心肌組織透射電子顯微照片(Transimission electronic micrograph, TEM)顯示提示心肌細胞核退行性變,核周線粒體減少或消失,肌絲部分溶解等,其余組則未見上述改變。 結論：新型阿霉素前體藥PDOX純度高、理化性質優(yōu)良。在CRS+HIPEC基礎上,聯(lián)合PDOX可進一步延長荷瘤動物生存期,安全性好。與DOX比,PDOX具有高效低毒的特點。PDOX具有進一步開發(fā)的價值。
[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.
緇撴灉錛氬悎鎴愮殑PDOX涓虹孩鑹插浐浣，

本文編號：1429253