载IκBα-siRNA纳米微粒靶向调控恒河猴眼小梁网及葡萄膜巩膜房水流出通道的研究

By means of increasing aqueous humor outflow, decreasing intraocular pressure (IOP) is the main strategy of treating glaucoma. However, incompliance caused by frequently implication of antiglaucomatic eye drops and damage of ocular surface is the major risk factor of glaucomatous blindness. Our previous study has confirmed that IκBα-siRNA will activate MMPs, resulting in IOP decrease. On this basis, we use sustained-release nanoparticles which are synthesized previously with hyperbranched glycogen derivatives (Glyp), IκBα-SiRNA and L-PLA-PEG copolymer to conjugate with single-chain antibody of trabecular meshwork and ciliary muscle cell. With intracamerally transferred immune-targeting nanoparticles, trabecular meshwork and ciliary muscle of normal and laser-induced glaucomatous rhesus monkeys are observed in vivo and in vitro to assess the transfection efficiency, sustained release capability and safety of the nanoparticles. By dynamically detecting IOP, retinal nerve fiber layer thickness, and expression of MMPs, TIMPs and ECM in trabecular meshwork and ciliary muscle, we hope to clarify the long-term regulation effect of this nanoparticles which is targeting trabecular meshwork and uveoscleral outflow pathway. We also hope to seek IκBα as a probable target for decreasing IOP in glaucoma, aiming to establish the foundation for new, persistent, safe and potent glaucoma treatment method.

促进房水外流降低眼压是青光眼治疗的主要策略，而降眼压药物频繁使用和眼表损害使患者依从性下降是青光眼盲的重要原因。我们前期已证实IκBα-siRNA可增强MMPs活性，降低眼压，在此基础上本项目用已合成的超支化糖原衍生物（Glyp）载IκBα-siRNA纳米微粒，连接可增强缓释性能的L-PLA-PEG共聚物，分别与抗小梁细胞及睫状肌细胞特异性抗原的单链抗体偶联，制备免疫靶向纳米微粒，通过前房注射转染正常恒河猴及激光诱导青光眼模型小梁网及睫状肌，从细胞和动物水平考察该纳米微粒的转染效率、缓释性能及安全性，检测小梁网及葡萄膜巩膜房水流出通道中MMPs、TIMPs和ECM的表达，动态监测动物眼压，测量视网膜神经纤维层厚度，从而明确载IκBα-siRNA免疫靶向纳米微粒在房水流出双通道中的长效调控作用，探讨IκBα作为降低青光眼眼压分子靶点可能性，为寻求作用持久、安全强效的青光眼治疗新策略打下基础。

促进房水外流降低眼压是青光眼治疗的主要策略，目前降眼压药物的频繁滴用和眼表损害使患者依从性下降是青光眼盲的重要原因。本课题通过制备超支化糖原衍生物（Glyp）负载IκBα-siRNA纳米微粒，从大鼠和食蟹猴体内外实验观察该纳米微粒的转染效率、缓释性能及安全性，寻求新的、更为安全有效的降低青光眼眼压的方法。结果如下：成功制备出Glyp负载IκBα-siRNA纳米微粒并验证其生物学性能及其于体内外实验的有效性。通过Real Time-PCR及western blot证实食蟹猴睫状肌及小梁网细胞中Lipofectamine® RNAiMAX介导的IκBα-siRNA 在mRNA及蛋白表达水平对IκBα具有抑制作用，同时促进MMPs mRNA及蛋白表达，抑制TIMPs mRNA及蛋白表达。在大鼠体内实验，Glyp介导IκBα-siRNA优化转染条件RNAi后，IκBα mRNA及蛋白表达量均受到明显抑制; RNAi后各时间点NF-κBp65 mRNA 及蛋白表达量变化无显著性差异，而免疫荧光检测显示NF-κBp65发生了核转位; MMPs mRNA表达量及酶活性均在RNAi后增加; Glyp介导的IκBα-siRNA在转染后1-5d在大鼠睫状肌Cy3荧光强度未见明显减弱;转染后2-4天，IV型胶原含量下降，同期眼压较基线眼压下降；RNAi后 7d及14d，大鼠眼球无眼前段并发症，且睫状肌组织HE染色也未观察到睫状肌组织结构及前房明显炎性细胞浸润及病理损害。再行食蟹猴体内实验，分别于术后24h及72h取眼球制作冰冻切片，在显微镜下观察发现Cy3红色荧光分布于小梁网及睫状肌，并再次验证IκBα-siRNA的降眼压作用。通过本研究证实了IκBα/NF-κB对葡萄膜巩膜及小梁网房水流出双通道中 MMPs的调控作用，在大鼠及食蟹猴体内外实验中下调IκBα表达，可激活NF-κB信号传导通路，增加MMPs、降低TIMPs的表达，减少胶原含量，最终降低眼压。Glyp作为 siRNA 转染的有效载体，具有极低的毒性、较好的生物相容性及较高基因转染效率。Glyp负载IκBα-siRNA在实验动物房水流出双通道中具有强效、安全和较持久的调控作用，有可能成为今后青光眼治疗的新靶点，为青光眼治疗新策略提供较好的实验依据。