蜂毒素抑制乳腺癌骨转移及溶骨性骨破坏机制的研究

With a high incidence rate and enormous harmfulness, metastatic bone tumors are lack of effective treatments. It is very meaningful to develop a new kind of drugs that can inhibit bone metastasis. Our group has been studying on melittin for several years. Our preliminary studies showed that melittin can inhibit the growth of osteosarcoma, and can reduce the expression of CXCR4 and TGF-β which are related to osteolytic bone destruction. Metastatic bone tumors and osteosarcoma have certain similarities: a high degree of malignancy; are bone isotropic; may lead to bone destruction. Therefore, we assume that, since melittin can inhibit osteosarcoma cells in vitra, inhibit rat osteosarcoma’s growth, then does it play the same role on metastatic bone tumors? The results we got from preliminary experiments showed that melittin could inhibit the proliferation and migration of breast cancer cells with high tendency of bone metastasis. In this study, we will establish a breast cancer bone metastasis model by injecting breast cancer cells into left ventricular, observe the inhibitive effect of melittin on metastatic bone tumors deriving from breast cancer ,and use the combined methods both in vitro and in vivo to detect the inhibitive effect of melittin on osteoclasts and the protein expression of cytokines (CXCR4, TGF- β, IL-11, PTHrP, VEGF) closely related to osteolytic bone destruction. Our study, for the first time to observe the inhibitive effect of melittin on metastatic bone tumor and to explore the mechanism of this action. This study will provide experimental evidence for the expansion of the scope of application of melittin in anti-tumor treatment, it will also provide experimental evidence for the development of a new drug for preventing and treating metastatic bone tumors.

转移性骨肿瘤发病率高，危害性大，临床缺乏有效的治疗措施。我们对蜂毒素的研究已多年，前期研究表明蜂毒素体内外均能够抑制骨肉瘤生长，并能降低与溶骨性骨破坏密切相关的CXCR4、TGF-β表达。转移性骨肿瘤与骨肉瘤具有以下共性：恶性程度高；均为骨向性；均可导致骨质破坏。因此，我们设想，蜂毒素既然能够杀伤体外培养的骨肉瘤细胞，抑制大鼠骨肉瘤生长，那么它对转移性骨肿瘤是否也存在着相似的作用？在预初实验证明蜂毒素能够抑制乳腺癌高骨转移细胞株增殖及迁移的前提下，本实验拟通过左心室注射法建立乳腺癌骨转移模型，观察蜂毒素对乳腺癌骨转移的抑制作用，体内外观察蜂毒素对破骨细胞及与骨转移瘤溶骨性骨破坏密切相关的细胞因子（CXCR4、TGF-β、IL-11、PTHrP、VEGF）蛋白表达的影响，从抑制溶骨性骨破坏角度探讨蜂毒素抑制转移性骨肿瘤的机制。为扩大蜂毒素的临床应用，开发防治转移性骨肿瘤的药物提供实验依据。

【研究背景】转移性骨肿瘤发病率高，危害性大，临床缺乏有效的治疗措施。我们对蜂毒素的研究已多年，前期研究表明蜂毒素体内外均能够抑制骨肉瘤生长，并能降低与溶骨性骨破坏密切相关的CXCR4、TGF-β表达。转移性骨肿瘤与骨肉瘤具有以下共性：恶性程度高；均为骨向性；均可导致骨质破坏。因此，我们设想，蜂毒素既然能够杀伤体外培养的骨肉瘤细胞，抑制大鼠骨肉瘤生长，那么它对转移性骨肿瘤是否也存在着相似的作用？为验证此假设，我们设计了此课题。.【研究内容】MTT法观察蜂毒素对MDA-MB-231细胞的增殖抑制作用，划痕闭合实验及transwell小室趋化实验观察蜂毒素对MDA-MB-231细胞迁移能力的影响，再通过胫骨骨髓腔注射法建立乳腺癌骨转移裸鼠模型，予蜂毒素干预，计算抑瘤率，从而可以得出蜂毒素是否抑制转移性骨肿瘤的结论。在确定蜂毒素对转移性骨肿瘤具有抑制作用的前提下，进一步从抑制溶骨性骨破坏角度探讨其可能的作用机制。采用MTT法观察蜂毒素对破骨细胞的直接抑制作用，Western Blot法观察蜂毒素对MDA-MB-231细胞分泌的与骨转移瘤溶骨性骨破坏密切相关的细胞因子（TGF-β、IL-11、PTHrP、VEGF）蛋白表达的影响，通过TRAP staining观察蜂毒素对骨转移瘤骨－肿瘤界面破骨细胞数目的影响，免疫组织化学法及Western Blot法观察蜂毒素对骨转移瘤组织中溶骨性骨破坏密切相关的细胞因子（TGF-β、IL-11、PTHrP、VEGF、CXCR4）蛋白表达的影响，从而得出验证假设的结论。.【研究结果】蜂毒素体外明显抑制乳腺癌MDA-MB-231细胞增殖及迁移，体内抑制移植瘤的生长，蜂毒素体外直接抑制破骨细胞增殖，体内减少移植瘤与骨界面破骨细胞的数量，蜂毒素体内外能够降低移植瘤及MDA-MB-231细胞中TGF-β、IL-11、PTHrP、VEGF、 CXCR4蛋白的表达。.【研究结论】蜂毒素存在着明确的抑制乳腺癌MDA-MB-231细胞增殖及抑制裸鼠胫骨移植瘤生长的作用，此作用可能与直接抑制破骨细胞增殖及降低溶骨性骨破坏相关因子（CXCR4、TGF-β、IL-11、PTHrP、VEGF）蛋白的表达有关。.【科学意义】该发现为扩大蜂毒素抗肿瘤的应用范围，开发防治转移性骨肿瘤的药物提供了实验依据。