超声影像介导Peri-Gem-Cal多功能纳米载体调控胰腺癌间质微环境的实验研究

It has been clearly proved that the adjuvant therapy of targeting pancreatic cancer cells cannot increase the five-year survival rate for pancreatic cancer patients. The newly developed regime of increasing the effect of adjuvant therapy in pancreatic cancer patients was regulating stromal compartments. The core idea of this strategy is not only controllable delivery of chemotherapy drugs but also balancing the pros and cons effect of stromal towards unto cancer cells. The domestic and international research nowadays in progress was focused on realizing both those ideas. In previous studies, the applicant research group had developed an advanced drug delivery system, which was mediated by coupling ultrasound and Nano-particles. This coupling method had achieved the effects of drug accumulation, tumor inhibition and visualized delivery under ultrasound in vitro and in vivo, and was regarded as an integration of diagnosis and treatment, which is expected to supply a comparatively well-equipped strategy in remodeling stromal compartment in pancreatic cancer patients. In this study, the applicant proposed to prepare a new multifunctional Nano-capsule, which includes the Gemcitabine (Gem) and Calcitrol (the analogues of Vitamin D which can “rest” the stromal) and was modified with Periostin on its surface. The Nano-capsule will be accumulated locally in pancreatic cancer stromal by the guidance of specific targeted antibody and the irradiation of ultrasound. The Nano-capsules will be visualized by ultrasound, and the changes of stromal elements will be estimated by quantification analysis, which will control the delivery process. Taking the advantage of Calcitrol in “resting” pancreatic stromal, the cell-killing effect of Gemcitabine will be increased, and the goal of extending the 5-year survival rate of pancreatic cancer patients is expected to be realized.

单独针对胰腺癌细胞的靶向疗法已证实难以改善胰腺癌患者五年生存率，调控间质组织是最新倡导的发展方向，其核心理念是可控性递送化疗药及稳定间质组织动态平衡，未来研究以两者为焦点。课题组前期研发的超声靶向递送纳米技术在胰腺癌细胞及体内移植瘤上实现了药物富集、抑制率增强及可视化递送的效果，是一种诊疗一体化的靶向给药策略，有望为调控间质组织提供一种较为齐备的治疗模式。本课题拟制备包载吉西他滨Gem和维生素D类似物卡泊三淳Cal的纳米微囊，其表面修饰靶向识别胰腺间质组织骨膜蛋白Per的特异抗体；在靶向抗体的引导及超声辐照下有效促进复合纳米载体Peri-Gem-Cal进入胰腺癌间质组织，依据超声图像量化微囊来评估治疗前后胰腺癌间质成分的改变，从而进行调控性给药，利用卡铂三淳“静息”间质组织活跃的优势，使吉西他滨更好地发挥对胰腺癌细胞杀伤作用，从而最终实现提高化疗疗效延长患者生存期的效果。

Periostin基因在胰腺癌中的表达量是正常胰腺组织的42倍，然而，Periostin基因在胰腺癌发生发展中的具体作用机制迄今缺乏系统性研究。为了探明Periostin基因与胰腺癌发生发展以及治疗中的确切关系，本课题组开展了如下几方面的研究：（1）Periostin基因在胰腺癌进展中的功能研究。（2）Periostin基因对胰腺癌血管生成的影响。（3）Periostin基因与胰腺癌化疗敏感性的关系研究。利用qRT-PCR，Western blot等技术检测Periostin基因在胰腺癌细胞，胰腺导管上皮细胞，胰腺间质细胞，胰腺癌组织及其匹配的癌旁组织中的表达情况。应用组织芯片分析Periostin基因在胰腺癌患者中的表达，及其与临床病理特征和预后的关系。通过超声靶向破坏微泡（UTMD）技术在胰腺星形细胞（PSCs）中转染慢病毒载体（Periostin-shRNA1, Periostin-shRNA2，Periostin-Control shRNA）沉默Periostin基因或利用人重组Periostin蛋白（rPeriostin）使Periostin基因在胰腺癌细胞中高表达后进行体内和体外实验。Periostin基因仅在胰腺间质细胞（PSCs）以及胰腺癌间质组织中特异性表达，在胰腺癌上皮细胞以及胰腺癌实质中不表达。另外，我们利用UTMD技术提高了胰腺间质细胞PSCs的慢病毒的转染效率，具有广泛的应用前景，同时为临床胰腺癌基因治疗提供了一定的指导意义。体内外实验结果证实Periostin基因可以促进胰腺癌细胞的增殖、迁移、侵袭、克隆形成以及小管形成能力，而且沉默Periostin基因后抑制了裸鼠成瘤能力，使瘤体的生长速度和大小均减慢，血流也明显减少。此外，沉默Periostin基因后提高了吉西他滨对胰腺癌细胞的杀伤力。 在机制研究中，我们阐明Periostin基因通过激活EGFR-Akt和EGFR-Erk-c-Myc信号通路来调节胰腺癌细胞的生存，转移和基因表达，通过Erk/VEGF信号通路来促进胰腺癌的血管生成，且通过调节Akt和Erk信号通路来介导对吉西他滨的化疗抵抗。Periostin基因在胰腺癌间质中高表达，并与淋巴结转移（p<0.01）和pTNM分期（p<0.05）有关，分期越晚Periostin基因表达越高。