特异性靶向纳米载体联合AQP1-siRNA对子宫内膜异位症的作用及其机制研究

The etiology and pathogenesis of endometriosis is still unclear which result in the lack of clinical treatment countermeasures. Biological treatment is a research hotspot in recent years. The expression of AQP1 in endometriosis cells and related signaling pathways can be silent and blocked by using the specific small interfering RNA, which play the effect of treatment of endometriosis. But the key is how to put the siRNA into endometriosis cells. In this project, we design the organelle-targeted non-viral vectors with the high efficiency and low toxicity as the goal. The drug delivery system of the AQP1-siRNA mediated with the nanoparticle was studied for intracellular transport and subcellular targeting, which reduced the cellular uptake of macrophage and normal cells, extended circulation time in vivo, and improved the endometriosis tissue distribution. The expression level of AQP1 in ovaries, uterus and lesion was examined. The lesion size, weight, VEGF expression, vascular density and apoptosis index, blood hormone levels, and ovarian and changes in uterine morphology were studied and AQP1-siRNA in the treatment of endometriosis with the process of signaling pathways was analysised. Then study the mechanism of AQP1-siRNA gene therapy and explore the feasibility of the siRNA gene therapy on endometriosis. And explore new theories, new directions and new ways for the treatment of endometriosis, which enjoying important theoretical and technological innovation, vision for the clinical treatment of endometriosis disease in which provides a new approach to development.

子宫内膜异位症的病因及发病机制不明，导致临床治疗对策缺乏。生物治疗是近几年的研究热点。采用特异性小分子干扰RNA，可沉默并阻断内异细胞AQP1的表达及相关信号途径，从而起到治疗内异症的效果，但siRNA对内异细胞的导入是关键。本项目以基因载体的高效、低毒为目标，采用具有细胞器靶向功能的非病毒载体，研究其介导siRNA给药系统的细胞内转运和亚细胞器靶向，减少巨噬细胞和正常细胞的摄取，延长体内循环时间，提高内异组织分布；考察卵巢和子宫以及病灶AQP1的表达水平，病灶体积、重量、VEGF表达水平、血管密度及细胞凋亡指数，血液性激素水平变化，以及卵巢和子宫的组织形态改变并分析AQP1-siRNA在治疗内异症过程中的信号通路；探索新型靶向纳米载体介导AQP1-siRNA治疗内异症的有效性与安全性，并研究其作用机制，为最终用于临床治疗内异症，提供新理论、新方法和新途径。

子宫内膜异位症的发病机制目前尚不完全清楚，临床上缺乏副作用小且高效的治疗药物。我们研究设计了具有特异性靶向功能的给药系统可成功把AQP1-siRNA 基因转运至靶组织，并能干扰沉默内异血管内皮细胞中过度表达的AQP1基因，用于子宫内膜异位症的治疗。研究发现，当靶向载体（CSO-PEI）/HA与siRNA氮/磷比为4时，制备得到的基因给药系统平均粒径为149.1nm，原子粒显微镜发现该形态为椭圆球或球型，可保护siRNA免受核酸酶攻击，保证了在细胞转染时AQP1-siRNA 的有效性。MTT细胞实验发现该基因给药系统细胞毒性低，具有HA的载体可快速进入子宫内膜癌细胞。通过尾静脉注射给药的方式将AQP1-siRNA 基因给药传输系统作用于建成的SD大鼠子宫内膜异位症模型，发现大鼠的子宫内膜异位症病灶明显缩小。AQP1-siRNA促进了异位内膜细胞的凋亡。经治疗后的内异组织内CD44表达减少。在电镜下观察发现大鼠在药物治疗后，其子宫、卵巢形态在用药组细胞及细胞器形态规则，未见核碎裂、边聚等凋亡形态，亦未见凋亡小体。进一步的研究发现，经siRNA治疗的内异病灶在SMA标记的成熟血管的微血管密度上无差异，而vWF标记的新生血管的微血管密度则明显减少。因此，该靶向载体介导的siRNA治疗可作为一种有效、安全的方法应用于内异症的治疗。同时研究发现该载体在肿瘤治疗方面也有突出表现。

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