超声分子靶向技术在体评价乳腺癌SDF-1表达及其促血管形成机制的研究

Tumor growth, invasion, and metastasis depend on the new blood vessel formation and tumor recurrence and metastasis is closely related to the tumor stem cells , previous studies have confirmed that Stromal cell-derived factor-1（SDF-1）plays an important role in endothelial cells or endothelial progenitor cells (EPCs) participating in the formation of blood vessels through its receptor CXCR4, but the newly research showed CXCR7 is another receptor of SDF - 1, and highly expressed in tumor vascular endothelial cells. Therefore, in the experiments, combining with the current ultrasonic imaging technology targeted molecule, to put forward that by targeted SDF - 1 ultrasound contrast agents, SDF - 1 expression is evaluated in breast cancer stem cell transplantation tumor, and futher explore wether SDF-1 is correlated with microvascular density, the number of endothelial progenitor cells and CXCR7 expression in the transplant tumor, thereby to provide a new method for clinical noninvasive assessment chemokine SDF - 1 expression in tumor; In addition, SDF - 1 / CXCR7 axis is further studied by using EPCs exposed to culture supernatant of transfected SDF - 1 breast cancer stem cell.

肿瘤生长、侵袭和转移依赖于新生血管的生成，肿瘤的复发和转移还与肿瘤干细胞存在密切相关，以往的研究证实SDF-1在内皮细胞或内皮祖细胞参与的血管形成中起重要作用，且通过CXCR4发挥作用，但新近发现CXCR7是SDF-1的另一作用受体，且高表达于肿瘤血管的内皮细胞。因此，在实验中，结合当前超声靶向分子成像技术，提出采用SDF-1靶向超声造影剂在体评估乳腺癌干细胞移植瘤内SDF-1表达，探讨其与移植瘤内微血管密度、内皮祖细胞数量及CXCR7表达的相关性，为临床在体无创评估肿瘤内趋化因子SDF-1表达水平提供一种新的方法；此外，利用转染SDF-1的乳腺癌干细胞培养上清作用的内皮祖细胞进一步研究了SDF-1/CXCR7轴对内皮祖细胞的影响。

研究表明，SDF-1/CRCX7生物学轴在肿瘤的发生、发展和转移过程中发挥重要作用，但SDF-1/CXCR7对乳腺癌侵袭转移的影响以及是否参与维持乳腺癌干细胞特性尚不十分清楚。因此本研究通过建立SDF-1过表达的乳腺癌稳定转染细胞系，探讨SDF-1对乳腺癌细胞侵袭、迁移和上皮间质转化的影响及相关生物学机制；通过免疫磁珠分选获得乳腺癌干细胞，采用RNA干扰的方法下调CXCR7表达，探讨SDF-1/CXCR7生物轴在维持乳腺癌干细胞表型方面的作用及相关分子机制。课题通过体内、外实验验证：（1）建立SDF-1过表达的MCF-7稳定转染细胞系，验证SDF-1过表达对乳腺癌细胞增殖、侵袭、迁移能力、上皮间质转化及对乳腺癌肿瘤干细胞表型的影响。结果显示SDF-1过表达可通过激活NF-κB和Wnt/β-catenin信号通路诱导MCF-7细胞上皮间质转化，并可促进细胞增殖、侵袭、迁移，诱导乳腺癌肿瘤干细胞表型的分化。（2）采用免疫磁珠分选CD44+/CD24− MCF-7细胞获得乳腺癌肿瘤干细胞BCSCs，构建CXCR7干扰的BCSCs稳定转染细胞系验证CXCR7表达下调对BCSCs增殖及干细胞特性的影响。结果显示CXCR7表达下调抑制乳腺癌肿瘤干细的增殖及干细胞特性。（3）将稳定转染的NC-BCSC、CXCR7 shRNA-BCSC细胞注射于裸鼠皮下组织，并给予化疗药物表阿霉素干预，结果显示CXCR7表达下调抑制乳腺癌肿瘤干细胞体内肿瘤的形成并增强化疗药物的敏感性。（4）对上述各组裸鼠模型经尾静脉注射超声造影剂，发现CXCR7 shRNA联合表阿霉素组移植瘤内血流灌注较对照组减少，造影参数峰值强度之间存在差异趋势。（5）提取MCF-7、pEGFP-N1-vector、pEGFP-N1-SDF-1组乳腺癌细胞和NC-BCSC、CXCR7 shRNA-BCSC及MCF-7组细胞培养上清分别作用内皮祖细胞，结果显示高表达SDF-1组促进内皮祖细胞的增殖，成管，迁移能力，CXCR7 shRNA组抑制了及内皮祖细胞的增殖，成管，迁移能力，证实SDF-1/CXCR7生物轴参与肿瘤血管的生成。本项目的完成对于寻找新的治疗靶点，对于开发新的化疗药物以延长乳腺癌患者的生存期，具有重要的理论和实际意义。