基于去细胞化组织基质和KGF构建的KGF-ECM-HP温敏型水凝胶对子宫内膜损伤的修复作用及机制研究

Endometrium plays an important role in protecting intrauterine physiology and pregnancy function for women. Increasing chances of trauma and infection result in the endometrial repair disorders, which have become the leading cause for intrauterine adhesion (IUA), amenorrhea and infertility. Current strategy of endometrial repair focuses on early intervention to reduce scar formation, promoting the regeneration of damaged intima, and maintain a special three-dimensional structure of the uterus. However, there is few strategies with high safety and efficiency in clinical treatment. Based on our preliminary studies, this project uses a new synthetic material heparin - poloxamer (HP) to prepare a novel HP hydrogel with suitable phase transition, anticoagulant and controlled release behavior. Extracellular matrix (ECM) and keratinocyte growth factor (KGF) are contained in the hydrogel structure to form KGF-ECM-HP emperature-sensitive hydrogel. Combined with in situ injection administration, KGF-ECM-HP hydrogel can attach closely on the womb and form a three-dimensional supporting gel. With comprehensive evaluation system including modern cytology, pharmacology, imaging, pathology and molecular biology, the multiple repairment effects and mechanism of KGF-ECM-HP temperature-sensitive hydrogel for endometrial damage model animals will be investigated. This strategy will combine these techniques effectively --"Biological graft material", "biological factor", "targeted delivery system". The strategy will overcome the limitation of existing therapies and satisfy the ideal requirement of safe, efficient and specific treatment to realize multiple repair effects on endometrial damage. Through our study, a novel strategy with high efficiency and safety will be developed to improve the clinical technology protecting intrauterine physiology and pregnancy function for Chinese women.

子宫内膜是维持女性生理生殖功能的重要保障，日益增多的创伤或感染因素导致的子宫内膜损伤成为女性宫腔粘连、闭经和不孕的首要原因。子宫内膜修复治疗重点在于早期干预减少瘢痕形成、促进已损伤内膜的修复再生和维持子宫内特殊三维结构，目前尚缺乏一种安全高效的治疗策略。本项目自主合成肝素-泊洛沙姆新材料，联合具有组织修复再生活性的去细胞化组织基质ECM和促进内膜上皮细胞生长的KGF，构建KGF-ECM-HP温敏型水凝胶，原位注射到子宫，形成紧密贴合子宫内壁的三维支撑型凝胶，通过现代细胞学、药理学、影像学、病理学、分子生物学等综合评价方法，研究原位注射KGF-ECM-HP温敏型水凝胶对于子宫内膜损伤模型动物的治疗效果，阐释其作用机制。本项目将生物支架/移植材料、高效治疗因子、制剂新技术多学科技术融合，克服已有治疗方法的局限，实现安全、高效的子宫内膜损伤的修复再生，提高我国女性生理生殖功能的医疗保障技术水平。

子宫内膜是维持女性生理生殖功能的重要保障，日益增多的创伤或感染因素导致的子宫内膜损伤可导致月经异常、不孕甚至反复流产。子宫内膜修复治疗重点在于早期干预减少瘢痕形成、促进已损伤内膜的修复再生和维持子宫内特殊三维结构，目前尚缺乏一种安全高效的治疗策略。本项目自主合成肝素-泊洛沙姆新材料，联合子宫去细胞基质（ECM）和促进内膜上皮细胞生长的角质细胞生长因子（KGF），根据KGF对肝素的亲和性结合制剂新工艺，最终制得KGF-ECM-HP水凝胶。通过电镜观察、胶凝温度、相转变温度的测定等检测方法完成了对其生物性能的全面评价，确证项目组成功制备了具有一定的抗凝活性，对于生长因子类药物有较好的亲和力，且其温度可控相变性的原位注射型制剂。本项目选择用子宫去细胞化支架为供体，提取支架去细胞化组织基质ECM，通过HE染色、扫描电镜、基因组DNA含量分析等评价去细胞化程度。通过Masson染色、ELASA法等检测ECM中胶原蛋白、纤维蛋白等的保留情况，证明生物支架材料的无免疫原性、高生物活性。通过细胞实验，考察不同配比的KGF-ECM-HP水凝胶对EEC的毒性及生长增殖状态的影响，判断各组KGF-ECM-HP水凝胶对子宫内膜细胞应用的安全性及促生长作用的有效性，确定KGF-ECM-HP水凝胶的具有最佳应用条件。通过大鼠子宫内膜损伤模型来验证原位注射KGF-ECM-HP水凝胶对子宫内膜损伤的修复效果。HE、Masson染色以及透射电镜观察组织显微形态结构变化验证子宫内膜损伤动物模型的成功建立。随后，项目组利用超声灰阶影像、HE染色、免疫组化染色等证实KGF-ECM-HP水凝胶促进子宫内膜修复。最后，项目组还完成了对子宫内膜损伤修复治疗后雌鼠生殖能力的进行了评价，证实KGF-ECM-HP水凝胶提高了损伤子宫妊娠率。本项目克服已有治疗方法的局限，实现安全、高效的子宫内膜损伤的修复再生作用，得到的技术方案有助于提高我国女性生理生殖功能的医疗保障技术水平。