血管新生在肝泡球蚴侵润性生长中的作用机制研究

Alveolar echinococcosis (AE) is one of the most serious parasitic zoonoses, caused by the metacestode of Echinococcus multilocularis.In huamns,metacestode cells of E multilocularis proliferate in the liver,inducing slowly progressive,life-threatening tumorlike growths. The parasitic lesions may subsequently metastasize to lung,brain and other neighboring organs, operation treatment and medication therapeutic efficacy is poor,and its rols of invasive and metastasis keeps unclear.Studies have shown that angiogenesis is one of the key markers of malignant tumors , VEGF and CD34 plays an important role in tumor angiogenesis and invasive growth process.Our preliminary study found that CD34 and VEGF were overexpression in the endothelial cells of alveolar hydatid tissues in alveolar hydatid model in gerbils .We speculate that angiogenesis play an important role in the invasion growth process of Echinococcus multilocularis.We plan to establish in vitro co-culture model and animal models of E.m and then use VEGF inhibitors and CD34 inhibitors for therapy .To detect the vascular changes of hepatic hydatid animal model dynamiclly by Micro CT ,Preliminary study the role that angiogenesis played in echinococcus multilocularis invasive and impact of anti-angiogenic drugs on echinococcus multilocularis growth through the changes of the the expression levels of CD34 and VEGF.The aim of this study is to elaborate the role of agiogenesis in the invasive growth of alveolar echinococcosis by in vitro and in vivo experiments, and obtain a new way for the clinic conservative treatment of patients who missed the best timing of surgery or inoperable .

泡球蚴病是对人体危害极大的人畜共患寄生虫病，原发于肝脏，呈恶性浸润性生长，可继发肺、脑转移，手术及药物治疗效果差，其浸润性生长和转移的机制尚不明确。研究表明血管新生是恶性肿瘤的关键标志之一，VEGF和CD34在肿瘤新生血管形成及侵袭性生长过程中起着重要作用。前期研究对沙鼠肝泡球蚴组织行免疫组化染色分析发现VEGF和CD34在泡球蚴组织囊壁内皮细胞呈阳性表达，据此我们推测血管生成可能与泡球蚴侵袭性生长有关。本研究通过体外培养泡球蚴和建立泡球蚴沙鼠模型，Micro CT动态检测肝泡球蚴动物模型血管变化，外源性给予VEGF和CD34抑制剂，通过CD34和VEGF指标变化，初步探讨血管生成在泡球蚴侵袭性中的作用及抗血管生成药物对泡球蚴生长的影响，为临床上泡球蚴病晚期患者的保守治疗、以及手术、药物的辅助治疗提供一个新途径。

背景：传统技术对肝泡球蚴病治疗效果有限。基于血管新生作为影响肿瘤快速生长关键环节的理论，抑制参与肿瘤血管形成因子表达或干预相关靶点已成为新药研发的重要突破方向。大量科学研究已证实抑制VEGF（血管内皮生长因子）、MVD -CD34（微血管密度）、OPN（骨桥蛋白）等在肿瘤血管生成因子可初步达到控制肿瘤发展速度效果。研究内容：本课题以前期研究即VEGF、MVD-CD34在肝泡球蚴浸润生长中血管新生的意义为基础，通过体外实验（缺氧模型）、体内实验（泡球蚴沙鼠模型）运用抗血管生成药物（重组人血管内皮抑素，恩度）及RNA干扰技术方法抑制两种因子表达，观察原头节活性、称量囊湿重、HE染色、免疫组化、ELISA、Western-blot等方法对比观察血管生长速度及因子表达。关键数据：体外培养中恩度对原头节活性无明显影响，Western Blot法分析对照组、25μg/ml组、50μg/ml组、75μg/ml组、100μg/ml组VEGF/β- actin 条带灰度值比值分别为：1.41±0.22、1.09±0.17、0.72±0.07、0.31±0.04、0.20±0.03；CD34/β- actin条带灰度值比值分别为：1.01±0.21、0.77±0.15、0.56±0.18、0.51±0.09、0.42±0.07；动物实验中泡球蚴沙鼠模型注射5 mg/kg恩度组、10mg/kg恩度组、15mg/kg恩度组的抑囊率分别为11.5%、31.4%、35.7%；注射质粒实验中VEGF组、CD34组、联合组的抑囊率分别为25.03%、13.97%、35.70%，各组中VEGF/CD34免疫组化评分、MVD值及蛋白定量水平较对照组不同程度下降。结果：恩度抑制肝泡状棘球蚴原头节的VEGF、CD34表达、降低微血管密度，一定程度上抑制泡状棘球蚴的生长；VEGF shRNA、CD34 shRNA质粒均能下调沙鼠肝泡球蚴VEGF、CD34表达，可有效抑制其浸润性生长中血管新生，延缓肝泡球蚴生长速度，VEGF shRNA质粒对肝泡球蚴作用强度高于CD34质粒。科学意义：本实验以沙鼠泡球蚴模型为基础，通过药物抑制泡球蚴血管生长，初步探讨血管生成在泡球蚴侵袭性生长中的作用及抗血管生成药物对泡球蚴生长的影响，为临床上泡球蚴病晚期、手术后复发患者的药物辅助治疗提供一个新途径。