以hMSCs为靶标的纳米探针及多模态成像在肝纤维化治疗的研究

End-stage liver disease has poor clinic prognosis. Liver fibrosis is an important pathological change for chronic type B hepatitis developing into end-stage liver disease. Low differentiation efficiency, potential negative differentiation, and ambiguous therapeutic mechanism are unsettled problems in the treatment of liver fibrosis using hMSCs. hHGF enable hMSCs to differentiate into hepatocyte-like cells with high efficiency in fibrotic microenvironment. This proposal will focus on GD2, a novel surface marker for hMSCs identification, as target, and design and fabricate BLI/MRI multi-modality nano-probes. The localization and the number of hMSCs could be sensitively and accurately evaluated by the integration of BLI and MR imaging. Moreover, on the basis of the hMSCs-targeted molecular imaging, targeted gene modification and differentiation regulation of hMSCs will be implemented. The ultimate goal is to realize multi-dimensional, dynamic and quantitative monitoring of the whole biologic process of hMSCs in the fibrotic liver, including disposition, survival, proliferation, differentiation and differentiation regulation of hMSCs. The anticipant results will provide fresh theoretical basis and scientific foundation for the treatment of liver fibrosis using hMSCs.

终末期肝病的临床预后极差，肝纤维化是慢乙肝发展至终末期肝病的重要病理变化。人骨髓间充质干细胞（hMSCs）治疗肝纤维化存在分化效率低，潜在负性分化，治疗机制不清等问题。人肝细胞生长因子（hHGF）能够在肝纤维化的微环境中高效诱导hMSCs向肝样细胞分化。本课题拟以hMSCs新型表面标志物二唾液酸神经节苷脂GD2为靶点，制备高性能光学(BLI)/磁共振（MRI）多模态纳米探针，整合活体生物发光和磁共振成像的优势，以期实现对hMSCs分布位置的灵敏定位以及其数量的准确评估；在此影像平台的基础上，进一步实现对hMSCs的靶向基因修饰和分化调控，以期实现多方位、动态和定量地靶向监测hMSCs肝内移植后的分布、存活、增殖、分化和分化调控的整个生物学过程。为hMSCs治疗肝纤维化提供新的理论基础和科学依据。

背景：人骨髓间充质干细胞（hMSCs）治疗肝纤维化存在分化效率低，潜在负性分化，治疗机制不清等问题。人肝细胞生长因子（hHGF）能够高效诱导hMSCs向肝样细胞分化。本课题制备高性能多模态纳米探针，实现多方位、动态和定量地靶向监测hMSCs肝内移植后的分布、存活、增殖、分化和分化调控的整个生物学过程。为hMSCs治疗肝纤维化提供新的理论基础和科学依据。方法：以hMSCs新型表面标志物二唾液酸神经节苷脂GD2为靶点，根据我们先前报道的方法合成scAbGD2-PEG-g-PEI-SPION和PAI/SPION两种纳米载体。采用凝胶阻滞实验评估其复合外源性基因的能力；动态光散射法测量scAbGD2-PEG-g-PEI-SPION/pDNA和PAI/SPION/pDNA纳米复合物的粒径大小及表面电位；体外细胞毒性实验检测其细胞毒性。使用流式细胞分析仪检测转染效率。通过激光共聚焦显微镜及普鲁士蓝染色实验研究BMSCs对纳米复合物的摄入。scAbGD2-PEG-g-PEI-SPION/pDNA和PAI/SPION/pDNA的光学及磁共振显像功能通过体外细胞和活体光学成像及磁共振扫描验证。结果：scAbGD2-PEG-g-PEI-SPION和PAI/SPION细胞毒性小，复合外源性基因后能够形成稳定的纳米复合物，粒径75-100 nm。在相同的N/P比值下，GD2靶向组的转染率明显高于非靶向组，抗体竞争抑制组和同型抗体组（P＜0.01）。N/P=20时靶向组取得最高转染率59.60 ± 4.50%。同时，scAbGD2-PEG-g-PEI-SPION中的SPION可有效标记hBMSCs，在磁共振T2/T2*加权图像上呈低信号。在肝纤维化小鼠肝脏冰冻切片中，与非靶向组相比，靶向组观察到更多的报告基因表达。在N/P=12时，PAI/SPION/pDNA转染大鼠间充质干细胞（rBMSCs）的转染率最高值70.2%±2.1%。PAI/SPION/pDNA转染的rBMSCs在大鼠体内光学成像和磁共振成像的时间达到移植后12天。实验大鼠的肝功能得到明显改善。结论：scAbGD2-PEG-g-PEI-SPION是一种磁共振可视的，能有效转染hBMSCs的靶向纳米基因载体。PAI/SPION能够有效转染rBMSCs。骨髓间充质干细胞肝内移植能够改善纤维化肝脏的功能。