仿生自组装KGF基纳米胶囊预防术后腹腔粘连的试验研究

Postoperative intra-abdominal adhesion (PIAA) is a universal complication that frequently occurs in patients who undergo abdominal surgery. There is still no reliable treatment to PIAA. Thus, developing a reasonable method to prevent PIAA would be most desirable from both fundamental and practical points of view. Based on the former research, we used KGF to promote the orderly repair of the mesothelial cell layer and thereby prevent the formation of peritoneal adhesions.In order to improve practical effect, two major problems need to be solved: First, the formulation of KGF needs to be improved to maintain the pharmacological efficacy of this agent in vivo; Second, how to keep peritoneum separated before it get repaired. By means of the cross discipline between medical science and advanced materials, we plan to prepare a self-assembly nanometer microcapsule which use biomimetic mussel adhesive protein, with polydopamine at the core, and with KGF binding on the functional group.It has the characteristics of enzyme-responsive and acid-responsive. The local inflammatory microenvironment of injured peritoneum and enzyme release with lactic acid formation which caused by hypoxia, can stimulate the microcapsule to release KGF. Besides, the colloid properties of it can physical isolate the injured peritoneal surfaces.Our study is divided into five stages: preparation of nanometer microcapsule, observation of physicochemical characterization, establishment of animal models and in vitro 3D tissue/cell culture. Adopting new techniques of nano-science, analytical chemistry, physical chemistry, experimental surgery, histomorphology and molecular biology, we study on preventive effect of peritoneal adhesion which biomimetic self-assembly KGF based nanometer microcapsule can provide, meanwhile, providing new preventive strategy for peritoneal adhesion.

腹腔粘连是腹部手术常见并发症，尚无可靠药物。寻找理想的预防方法仍是当前需解决的问题。课题组在前期实验基础上，发现角质细胞生长因子(KGF)可通过促进间皮再生预防粘连。为提高实际效果，需要解决两大问题：其一，如何维持KGF在体内微环境下持续的药理作用；其二，如何在受损腹膜修复之前保持分离状态。我们拟通过医学-先进材料交叉，制备一种贻贝粘附蛋白仿生、以聚多巴胺为核心、官能团结合KGF的自组装纳米微胶囊。其具有酶响应和酸响应特征，因受损腹膜微环境局部炎症和缺氧导致酶释放和乳酸生成，可刺激微胶囊缓释表面KGF。此外，它具有胶体特性，可以物理隔离相对的受损腹膜面。我们将在纳米微胶囊制备、理化表征观察、动物模型、体外3D组织细胞培养五个阶段，采用纳米材料学、分析化学、物理化学、实验外科学、组织形态学、分子生物学等方面技术，探究仿生自组装KGF基纳米微胶囊对腹腔粘连的预防效果，提供新的腹腔粘连预防策略。

腹膜受损时，间皮细胞的修复时机和速度对腹腔粘连的预防至关重要， KGF对间皮细胞有潜在的有丝分裂原活性,可促进细胞增殖。间皮细胞修复过程需要持续8天，腹腔内给予的KGF在体内各种生物酶等作用下，不能保持长时间的活性。因此，进一步改善剂型以维持KGF在体内的稳定持续的药理作用，是提高粘连预防效果的需要。我们推测：制备一种以聚多巴胺为核心、官能团结合KGF的自组装纳米微胶囊，在受损腹膜微环境局部炎症和缺氧导致酶释放和乳酸生成引起的酸性刺激下，缓慢的持续释放KGF，加速腹膜的间皮细胞恢复完整性，同时使用屏障类材料如透明质酸钠凝胶（Ha）保持腹膜受损面的分离状态，会更好的预防粘连形成。为证实该假说，我们（1）通过仿生技术与界面工程自组装构建自组装KGF基纳米胶囊，应用投射电子显微镜、扫描电子显微镜、动态光散射分析观察微纳米聚多巴胺(PDA MNPs)和微纳米聚多巴胺-KGF纳米粒子(PDA-KGF NPs)材料微观结构、孔直径和孔尺寸，应用X射线光电子能谱技术、红外光谱分析检测PDA MNPs和PDA-KGF NPs材料的表面官能团，元素含量及价态变化，应用流变仪分析、cck-8试验、EdU增殖分析、酶联免疫检测分析PDA MNPs和PDA-KGF NPs的流变特性、生物相容性、稳定性以及其酸响应缓释性能，结果显示PDA-KGF NPs制备成功，且具有理想的流变性能，生物相容性及生物稳定性；（2）通过建立腹腔粘连大鼠模型，取损伤部位壁层腹膜以及腹膜下组织给予不同干预措施并进行体外组织培养，于培养 48 小时后观察体外模型粘连带形成情况，结果揭示Ha联合PDA-KGF NPs在体外模型中可明显抑制腹腔粘连的形成；（3）通过动物实验研究，不同干预措施分别干预大鼠腹腔粘连模型并分别检测预定时间点大鼠一般生理状态，腹腔粘连程度，随后通过qRT-PCR、Western blot、免疫组化、天狼星红/HE染色等技术观察损伤腹膜间皮细胞层的修复情况，检测纤维化指标，炎症水平指标，炎症反应/纤维化相关分子通路，结果明确KGF 基纳米胶囊在体内具有预防术后腹腔粘连的作用，并且促进腹膜间皮细胞的修复的同时，可明显减少胶原沉积和纤维化，抑制炎症反应。本项目的成果揭示PDA-KGF NPs联合Ha作为新型屏障状生物材料可有效预防术后组织粘连发生，为预防术后腹腔粘连提供了一个有前景的临床方案。