【作者】 仲召阳；

【导师】 王东；

【作者基本信息】 第三军医大学 ， 病理学与病理生理学， 2005， 硕士

【摘要】 骨肉瘤是多发于青少年的富含血管的高度恶性肿瘤,其生长很大程度上依赖于血管的供应。目前疗效不佳,其中重要原因是瘤内存在伴发性肿瘤抵抗,而伴发性肿瘤抵抗和肿瘤血管生成特性相关。抗肿瘤血管生成治疗耐药性的获得可能与多种低氧反应性基因有关。DNA 损伤修复基因脱嘌呤/脱嘧啶核酸内切酶APE1 具有核酸内切酶及氧化还原双功能,系低氧诱导细胞凋亡的重要保护基因,推测与抗肿瘤血管生成的耐药性有关。RNA 干扰(RNAi)技术阻断基因表达具有高稳定、高效率和高特异的特点,完全有可能作为一种新的分子生物学工具成为肿瘤基因治疗的新手段。通过RNAi 技术抑制肿瘤DNA 损伤修复基因APE1 表达,增加肿瘤细胞抗血管生成敏感性,有效清除肿瘤细胞,是肿瘤基因治疗的一项重要策略。本项目应用免疫组化检测骨肉瘤中APE1 表达,分析APE1 与骨肉瘤演进以及血管生成的关系。并应用RNA 干扰技术,构建人体APE1 siRNA 表达载体pSilence APE1,“敲除”骨肉瘤细胞APE1 基因的表达。通过裸鼠荷瘤实验,荷瘤生长曲线观察、蛋白印迹和免疫组化等技术,探讨APE1“敲除”骨肉瘤细胞对抗血管生成治疗的反应。研究目的1、探讨DNA 损伤修复基因APE1 在骨肉瘤中的表达及其与肿瘤演进、血管生成及患者预后的关系。2、构建骨肉瘤APE1 siRNA 表达载体pSilence APE1,并检测其对HOS 和9901骨肉瘤细胞APE1 蛋白表达的敲除作用,同时探讨其在抑制内皮细胞迁移过程中的作用。3、通过肿瘤生长、病理组织学、免疫组化以及激光共聚焦等技术,观察pSilenced APE1 对ES 抗裸鼠骨肉瘤移植瘤血管生成协同作用。研究内容和方法1、APE1 蛋白在骨肉瘤细胞中的表达及其与血管生成的关系。免疫组化SP 法检测五种骨肉瘤细胞株(HOS、U-2OS、Sa-OS-2、9901、9706)、10 例正常骨、10 例良性骨瘤和60 例骨肉瘤组织中APE1 蛋白表达,并根据CD34 和F更多还原

【Abstract】 Osteosarcoma(OS) is , in general, a hypervascular tumor usually occurs in the second and third decade of life , the growth of which is largely dependent on the extent of blood supply. OS is so far a hard-to-cure diease, to which concomitant tumor resistance (CTR) may contribute much, and CTR is related with the characteristic of angiogenesis. The acquired resistance of antiangiogenesis may be associated with a lot of hypoxia-response genes. The human apurinic/apyrimidinic endonuclease(APE1), a bifunctional redox factor/AP endonucleae, plays a crucial role to protect against cell death due to the hypoxia. So we hypothesis that the Ape1 may be contribute to the resistance of anti-angiogenesis therapy. RNA interference (RNAi) may be a optimized molecular biological means with following important features: high stability, high efficiency, and high specificity. Hence, it may become a new means of tumor gene therapy. One strategy for tumor gene therapy is to inhibit expression of DNA damage and repair gene APE1, selectively enhance sensitivity of antiangiogenesis in osteosarcoma cells with the purpose of eliminating tumor cell rapidly and effectively. This study will investigate the expression of Apurinic/apyrimidinic endonuclease (APE1) protein in human osteosarcoma, also evaluate its role in tumor development and angiogenesis. Meanwhile, we constructed the specific Ape1 siRNA stable expression vector pSilence APE1 using the RNA interference technique, to "knock down" the APE1 expression in the osteosarcoma cell, and to determine the "knock down" cell response with anti-angiogensis treatment in the nude mice experiment by the growth curve observation, Western blot, as well as the microvascular count. This purpose is to enhance the tumor sensitivity to anti-angiogenetic therapy. Objective 1. To research the expression of APE1 protein in osteosarcoma cells and osteosarcoma cell lines, also evaluate its role in tumor development, angiogenesis and patient’s prognosis. 2. To construct APE1 siRNA expression vector pSilence APE1 and determine it’s function to "knock down" APE1 gene expression in osteosarcoma cell lines HOS and 9901, as well as its role to inhibit endothelial cell immegration induced by osteosarcoma cell. 3. To observe the synergistic role of pSilence APE1 with anti-angiogenesis treatment using Endostatin in tumor bearing nude mice by the growth curve observation, Western blot, as well as immunohistochemistry. Materials and Methods 1. The expression of APE1 protein in human osteosarcoma and its relationship with angiogenesis. Expression of APE1 proteins was detected immunohistochemically in 5 osteosarcoma cell lines(HOS、U-2OS、Sa-OS-2、9901、9706), 10 bone samples, 10 cases of human bone benign tumor and 60 cases of human osteosarcoma. Intratumor microvessel density(MVD) was counted according to the result of CD34 and FⅧ-RAg protein staining. 2. Construct APE1 siRNA expression vector and its inhibition of endothelial cell immigration. APE1 siRNA gene sequnces were screened, designed and synthsized by submitting GenBank and analyzing active domain sequnce of APE1, which annealed to formed ds-linker, inserted into linear expression vector and got pSilence APE1 siRNA. The vector was indentificated according to the DNA sequncing and double enzyme digestions. pSilencer APE1siRNA plasmid was transfected to 9901 and HOS cells by lipofectamine 2000. Dose-effect and time-effect relationship of APE1 gene silence inducing by RNAi were measured using Western blot analysis. Using EGFP expression plasmid, the transfection efficiency of RNAi vector was measured with fluorescence microscopy. Before and after transfection, immigration of endothelial cell was measured by Transwell. The change of proliferation, apoptosis was mansured using immunohistochemistry and TUNEL methods. 3. To study the effect of enhancing antiangiogenesis sensitivity of pSilence APE1 in OS with Endostatin therapy. Twenty nude mice bearing 20 grafted tumor(9901) were divided into four groups, POStreatment control, ES treatment only (1.5mg/kg, daily), psilence APE1 treatment only(20μg, twice/weekly) and combination ES and psilence APE1. The growth curve were measured, the histological changes were observed by H.E staining and microscopy, the Ape1expression, cell proliferation, MVD, hypoxia as well as apoptosis were determined by immunohistochemistry and TUNEL method. Results 1. The expression of APE1 protein in OS cells and its relationship with angiogenesis. APE1 protein was positive staining in five osteosarcoma cell lines and predominantly localized in the nucleus of osteosarcoma cells. The protein level of the APE1 in osteosarcoma was significantly high than that of bone and benign tumor. Forty-three of sixty osteosarcoma(72%) were determined to demonstrate high levels of APE1 expression, whereas the remaining 17 (28%) showed low expression of APE1. The degree of APE1 expression in osteosarcoma is related with prognosis. The MVD of the cases of APE1 high expression groups(46.4±26.7) was significantly higher than that of low expression groups(33.3±20.7)(P=0.03)。2. Construct APE1 siRNA expression vector and its inhibition of endothelial cell immigration. After evaluation and sequencing, OS expression vector pSilencer APE1 siRNA was constructed successfully, and transfected into 9901 and HOS cells by lipofectamine 2000. The result of western blot showed that, APE1 protein in OS cells could be koncked down specifically by pSilence APE1 siRNA vector, and inhibition rate of APE1 expression was 72-95%. The best inhibition of expression of APE1 gene was 3.0μg and at the 48 hours using pSilence APE1 siRNA vector. The results of EGFP plasmid was detected by laser cofocal scanning microscopy suggested that the transfected rate of RNAi plasmid was 28-62%. The condition of APE1 gene silence, induced endothelial cell immigration potential of HOS and 9901 cells was significantly decreased, and the apoptosis of 9901 and HOS cells was verified by TUNEL. The apoptosis rate in RNAi group was much higher than that of control group. The Proliferation index was significantly decreased by immunohistochemistry.3. To study the effect of enhancing antiangiogenesis sensitivity of pSilence APE1 in OS with Endostatin therapy. The tumor formation rate of 9901 osteosarcoma cell after injection in nude mice is 100 percent, the latent period is 3 days and growth steadily, which can provide material and model for further investigate of human osteosarcoma. Various dose of ES was given by intratumor injection. As a result, all tumor in nude mice were diminishing with time. The tumors of the therapy groups was much smaller than those of control group. Tumor-inhibition rate of low dose of ES and high dose of ES was 22.92% and 35.83%. The apoptosis index, the proliferation index and MVD were significant different in control group, low dose of ES group and high dose of ES group. The Tumor-inhibition rate of pSilence APE1 group, ES group and mixed group was 38.23%, 35.29% and 62.18% separately. The apoptosis index of various group was much higher than that of control group, while the proliferation index and MVD were much lower than that of control group. Furthermore, the apoptosis index of mixed group much higher than others and the proliferation index and MVD of mixed group was much lower than others, which is significnace of clinical combined anti-angiogenestic therapy in the future. Conclusion 1. The over expression of APE1 in osteosarcoma was significantly with tumor’s differentiation and tumor microvessel density. APE1 may be assiociate with angiogenesis and the gene theray targated with APE1 may be involved with improving antiangiogenesis treatment sensitivity. 2. APE1 gene in OS cells could be koncked down specifically by APE1 siRNA vector pSilence APE1. The largest inhibition effect of expression of APE1 gene was optimalized at dose 3.0μg and duration two days with pSilencerAPE1 siRNA vector. The efficiency of gene silence was 72-95% that induced by RNA interfencing technology in the reseach. 3. pSilence APE1 and ES can successfully inhibit endothelial cell immigration in HOS and 9901 ostesarcoma cell lines separately , furthermore, pSilence APE1 can potential improve the inhibition immigration of endothelial cell by ES. 4. Antiangiogenesis can efficiently inhibit osteosarcoma growth and proliferation,induce OS cell apoptosis, decrease the MVD of tumor. Antiangiogenesis could serve as an assistant therapy for OS. 5. Inhibiting APE1 expression of OS cells could improve antiangiogenesis sensitivity remarkably and induce apoptosis of OS cells, meanwhile inhibit the proliferation of OS cell and decrease MVD, which is significnace of clinical combined anti-angiogenestic therapy sensitivity in the future.更多还原