靶向CD133有机硅氟碳荧光纳米胶束在胰腺癌干细胞活体示踪及HIFU增效治疗中的实验研究

Pancreatic carcinoma was a high malignant disease with poor prognosis. Pancreatic cancer stem cell(CSC) and circulating tumor cells(CTCs) have close relationship with tumor metastasis and drug resistance. In our previous work, the CD133 antibody was connected with the organic silicon perfluorocarbon fluorescent nano micelle .It was also used as fluorescence molecular probe for pancreatic cancer diagnosis and therapy. By building the system, which consists of a small animal optical imaging system and an ultrasonic imaging detection system for living small animals, the pancreatic cancer stem cell was detected by bimodal imaging of fluorescence and ultrasonic imaging. Pancreatic tumors in nude mice were transfected to express green fluorescent protein (GFP),which was used as our animal model. In vivo flow cytometry(IVFC) was applied to monitor the pancreatic cancer CSC and CTCs dynamically. In this way,we combined the functional imaging and anatomic imaging together , also employed visual inspection and microcosmic test. And the aim to track the local growth ,metastasis and homing of pancreatic cancer CSC can come true. Meanwhile, perfluorooctyl bromide（PFOB）which was wrapped inside the fluorescence molecular probe can produce liquid gas phase shift and rupture under the function of ultrasound. Its characteristics can also make the HIFU therapy more accurately and enhance the efficacy of HIFU treatment. In conclusion, our study will provide a powerful research platform for the early track of pancreatic cancer CSC in vivo, and also offer a totally new idea of HIFU precise treatment for pancreatic cancer CSC.

胰腺癌恶性程度高、预后极差。胰腺癌干细胞（CSC）和循环肿瘤细胞（CTCs）与肿瘤转移和治疗的耐药性关系密切。本研究在前期工作中以靶向CD133有机硅氟碳荧光纳米胶束作为诊疗一体化荧光分子探针，通过所搭建的活体小动物光学成像系统及超声成像系统，对胰腺癌干细胞进行荧光/超声双模态成像；以绿色荧光蛋白（GFP）稳定表达的裸鼠胰腺癌作为动物模型，通过近红外双通道活体流式细胞仪对胰腺癌CSC和CTCs细胞进行动态监测，将功能成像和解剖成像相结合，宏观检测和微观检测相结合，实现对胰腺癌干细胞局部生长，转移及归巢的动态立体全面追踪。同时，靶向荧光纳米探针内部包裹的液态全氟辛溴（PFOB）可在超声作用下产生液气相转变并破裂，此物理靶向特点亦对HIFU治疗起到精确引导及增效作用。综上所述，本研究为胰腺癌干细胞早期活体示踪及HIFU精准治疗建立了良好的研究平台。

【研究背景】胰腺癌恶性程度高、预后极差。胰腺癌干细胞与循环肿瘤细胞与肿瘤转移及耐药性或关系密切。寻找针对胰腺癌干细胞的的特异性靶向系统及精确定位释放技术对于胰腺癌的精准治疗及改善预后有重要意义。.【研究内容与方法】采用原子转移自由基聚合（ATRP）等成熟的高分子聚合技术制备靶向CD133有机硅氟碳荧光纳米胶束作为荧光分子探针，检测其物理性质并评价其安全性。采用磁珠法分离稳定表达的胰腺癌干细胞体及裸鼠脊背胰腺癌皮翼视窗模型评价材料的体外及体内靶向性。制备裸鼠原位胰腺癌模型及皮下瘤模型，使用活体流式细胞仪检测外周血CSCs及CTCs，并通过超声及病理组织学及免疫组化检测评价HIFU联合靶向材料的消融效果。.【重要结果及关键数据】所制备的靶向CD133有机硅氟碳荧光纳米胶束呈球形，粒径尚均匀，平均粒径为100nm。Zeta电位为-3.6mv，其中PFH包裹率为15.2Wt%。低浓度材料对细胞没有毒性，高浓度时可抑制细胞的增殖，。胶束经HIFU辐照后有明显形变，直可增加4倍以上甚至破裂。使用免疫磁珠分选出CD133+荧光细胞，与CD133-细胞相比，细胞称悬浮球形生长，细胞排列紧密，以出芽方式增殖生长，且通过免疫印迹法检测CD133+中OCT-4蛋白较CD133-相比表达率明显增加，具有干细胞特性。激光共聚焦荧光显微镜下可以观察到靶向胶束与CD133+细胞的靶向性好，围绕在红色的细胞膜周围，具有良好的体外靶向性。制备裸鼠脊背胰腺癌皮翼视窗模型并于尾静脉注射材料后于双光子荧光显微镜下，可在同一切面观察到肿瘤组织与荧光材料的结合，即靶向材料有良好的体内靶向性。活体流式细胞仪可检测出原位胰腺癌模型中外周血循环肿瘤细胞，且在HIFU治疗后循环肿瘤细胞明显减少。靶向胶束联合HIFU消融胰腺癌皮下移植瘤病理组织学可见靶向胶束组凝固性坏死较对照组明显增多，细胞间隙增大，细胞核固缩，TUNEL染色消融灶内部分细胞发生凋亡，肿瘤组织内血管明显较少。.【科学意义】HIFU联合靶向CD133有机硅氟碳荧光纳米胶束治疗胰腺癌，将被动靶向、主动靶向及物理靶向作用相结合，为胰腺癌的活体示踪及HIFU精准治疗提供良好的诊疗一体化研究平台及奠定良好的动物实验基础。