【作者】 李伟栩；

【导师】 杨迪生；

【作者基本信息】 浙江大学 ， 外科学， 2002， 硕士

【摘要】 前言：虽然辅助化疗和新辅助化疗的应用，改善了骨肉瘤的预后，但高转移率仍是治愈该病的困难所在。传统化疗存在化疗耐药等问题，近年来抗肿瘤血管生成治疗研究迅速发展，虽然尚有许多潜在的问题待解决，但提供了一条有希望治愈肿瘤的新途径。抗肿瘤血管生成治疗的前景：(1)研究发现虽然在分子水平上肿瘤血管内皮细胞区别于正常内皮细胞，但各种肿瘤的血管内皮细胞是相似的。抗血管生成剂可有很广的抗瘤谱。(2)抗血管生成剂的作用靶点是内皮细胞，通常稳定、为二倍体，而不象肿瘤细胞易于突变形成耐药性。(3)化疗作用依赖于药物的渗透，对于少血供的肿瘤疗效就差。而血管内皮细胞易于接触血循环中的药物。(4)许多试验表明副作用可以接受，且与化疗药无毒性叠加，因为这类药为细胞稳定剂，而非细胞毒性剂。抗血管生成治疗基于以下策略：1)阻断血管增生因子、受体、或传导通路；2)上调或产生内源性抑制剂；3)直接作用于肿瘤血管。血管内皮生长因子(vascular endothelial growth factor，VEGF)、基质金属蛋白酶(matrix metalloproteinases，MMP)是抗血管生成治疗的两个重要靶因子。已有许多研究表明MMPs(尤其是MMP-2、MMP-9)、VEGF与肿瘤的恶性侵袭和转移有关。有关骨肉瘤的研究甚少，尚无在骨肉瘤中关于VEGF与MMP-2、MMP-9的相关性研究报道。 P．：罗t汐 一．…。，、．丛一ho 一 硕士学位论文 瓦s厂 目的：探讨骨肉瘤MMP－2、MMP－9与VEGF表达的相关性及其对预后的影响， 为抗血管生成治疗在骨肉瘤患者中应用提拱依据。 方法：采用兔疫组织化学技术对56例不同临床行为的骨肉瘤活检组织和8例转移 灶组织进行了VEGF、MMP－2、MMP－9表达的检测。本研究选用浙江大学医学院 附属H院 1994年 IR～2000年 12月期间收治的肢体＊B期骨肉瘤活检标本 56例， 并另有8例转移灶标本；其中男性34例、女性22例；年龄9—43 岁、中位年龄18 岁；股骨下端 28例、胚骨上端 12例、胖骨上端 5例、股骨上端 2例、肽骨上端 8例和腔骨下端1例：成骨细胞型28例、成软骨细胞型11例、成纤维细胞型8 例和其它类型9例；保肢37例，截肢19例。 免疫组化染色采用SP法，MMP－2、MMP－9仅作定性分析，大于5％细胞 阳染界定为阳性，＊＊OF作半定量分析，阳染率小于10％为阴性（－），＞10％且＜50％ 为弱阳性（＋），＞50％且＜80％为阳性（＋＋），Z80％为强阳性（＋＋＋）。用己知乳腺 癌转移淋巴结MMP－2染色阳性切片、乳腺癌转移淋巴结MMP－9染色阳性切片和 结肠癌VEGF染色阳性切片分别作MMPZ、MMP－9、VEGF的阳性对照；用 PBS分别代替第一抗体作阴性对照。SPSS 10．0统计软件作统计学处理。 结果：56例患者随访时间13～gi个月，中位时间47月；其中无瘤生存26例， 复发同时肺转移1例，软组织转移2例，骨转移1例，肺转移26例。三年无瘤生 存率50．3％，五年无瘤生存率38．8％。 l、MMP－2表达 MMP－2表达阳性的肿瘤细胞常聚集成团而成灶性分布，或分布于肿瘤灶的边。缘。MMP－2 表达阳性率为 53．6％；阳性组五年无瘤生存率（disease free ＊＊＊v1v＊1，OFS）为17．0％：阴性组五年OFS为69．2％。两组生存率经1。g－＊＊uk检 验有显著统计意义（p＝0．0012）。MMP－2表达与肿瘤大小经Kendall 等级相关 分析无统计意义。 2、MMP－9表达 2 pei罗i 4 一．．－ho 硕士学位论文 飞羹歹 MMPg表达阳性的肿瘤细胞分布与MMPZ类似；阳性率为53．6％；阳性组 五年无瘤生存率为23．9＄：阴性组五年DFS为59．8％。两组生存率经二。g－Y8llk检 验有显著统计意义（p＝0．0049）。MMP－9表达与肿瘤大小经 Kendal 等级相关分 析无统计意义。 3、VEGF表达 VEGF表达阴性卜）14例、弱阳性（＋）13例、阳性卜＋）14例、强阳性（＋＋＋）15 例。把阴性和弱阳性界定为低表达组，共27例，生存17例，五年DFS为57．5％； 阳性和强阳性界定为高表达式组，共29例，生存9例，五年DFS为24．l％。两组 生存率经 log－rank检验有统计意义（p＝0．0322）。 4、骨肉瘤原发灶与转移灶的MMP－2、MMP－9、VEGF表达 骨肉瘤转移灶中MMP－2、MMP－9、VEGF表达阳性率较高，且同一病例原发 灶与转移灶的表达有良好一致性。 5、MMPZ、MMPg与VEG更多还原

【Abstract】 IntroductionAlthough the use of adjuvant chemotherapy and neoadjuvant chemotherapy improves the prognosis of osteosarcoma, the high ratio of metastasis is still the problem. The traditional chemotherapy has many defects, such as drug resistance. Antiangiogenic therapy, at least in theory, provides a number of important advantages over cytotoxic chemotherapy: (1) Tumor endothelium appears to be similar to each other. This finding supports the notion that agents directed against tumor endothelium will be active for a broad range of tumor types, a hope that has been supported by animal studies. (2) Tumor cells lose responsiveness to chemotherapy in part because their high mutation rate and genetic instability lead to the selection of and overgrowth by resistant clones. Antiangiogenic therapy, by contrast, targets activated epithelial cells that are nonneoplastic, diploid, and genetically stable. This has lead to the prediction that resistance may develop more slowly, or not at all, to this type of treatment, which has been borne out in certain animal models. (3) The responsiveness of a tumor to chemotherapy is also dependent on tissue particularly in poorly vascularized, hypoxic tumors.Endothelial cell, however, should be easily accessible to agents administered systemically. (4) The side effect profile for many of the agents tested thus far appears to be favorable and is generally nonoverlapping with chemotherapies. The mechanisms of antiangiogenic therapy includes three aspects: (1) Block the cytokines for angiogenesis, receptors or signal pathways; (2) Up-regulate or induce endogenous inhibitors; (3) act directly on the blood vessels of the tumor. Vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMP) are important targets for anti-angiogenic therapy. Many studies has indicated that MMPs (especially MMP-2 and MMP-9) and VEGF are associated with tumor invasion and metastasis. However, there is no report concerning the correlation between VEGF, MMP-2, MMP-9 and osteosarcoma.AimThe aim of this study was to: (1) find the possible relationship between the expression of VEGF and the expression of MMP-2 and MMP-9 in osteosarcoma; (2) detect wether the expressions of MMP-2, MMP-9 and VEGF were associated with the prognosis of osteosarcoma; (3) provide evidence for antiangiogenic therapy in clinical use for osteosarcoma.MethodsThe 56 specimens of osteosarcoma biopsy and 8 specimens of metastasis focus were from the Department of Orthopaedics in the Second Affiliated Hospital of Zhejiang University. There were 34 specimens from male and 22 specimens from female. The age of the patients was from 9 to 43 years old and the mediumage was 18 years old. Tumors were located in the distal femur in 28 patients, the proximal tibia in 12, the proximal humerus in 8, the proximal fibula in 5, the proximal femur in 2 and distal tibia in one. The number of osteoblastic osteosarcoma is 28, chondroblastic 11, fibroblastic 8 and the others 9. Thirty-seven patients carried out limb salvage surgery, 19 patients carried out amputation.The expression of VEGF, MMP-2 and MMP-9 was detected by immunohistochemical method (S-P). The metastasis lymphonode of breast cancer was used for positive control of MMP-2 and MMP-9. The colon cancer was used for positive control of VEGF. MMP-2 and MMP-9 were described by qualitative analysis: "+" means positive cell >5%. VEGF was described by half-quantitative analysis: "-" means positive cell更多还原