载有缺氧诱导因子-1α小干扰RNA缓释系统的间充质干细胞对糖尿病刺激脉络膜新生血管的抑制作用

It is well known that ischemia and hypoxia caused by diabetes could lead to growth of pathologic retinal neovascularization, however, whether it stimulates formation and progress of choroidal neovascularization (CNV) has still been in debate. Previously, we found hyperglycemia can aggravate development of experimental CNV, meanwhile, bone marrow-derived stem cells (BMCs) contribute to formation of CNV by recruitment and incorporation in this progress. In addition, expression and activity of hypoxia-inducible factor 1α (HIF-1α) increase in response to low oxygen tension, which play critical roles in the process of CNV and diabetic retinopathy. Previously, we formulated hypoxia-inducible factor 1α short hairpin RNA plasmid DNA loaded poly (D,L-lactide-co-glycolide) combined with polyethylenimin nanoparticles (NPs), and intravitreal injection of this nanoparticle reduced the extent of CNV. However, tranditional methods of drug administration inevitably impact the physiologic function of retina and can not reach the goal of sustained release. Therefore, our study will explore a targeted therapeutic strategy of genetic engineered mesenchymal stem cells (MSCs) as delivery vehicles on CNV under hyperglycemia and hypoxia. Proliferation, migration and differentiation of MSCs were observed by immunofuorescence staining and in vivo optic imaging of animals after establishment of luciferase chimeric diabetic mice. Furthermore, effect of MSCs carrying NPs on laser induced CNV of diabetic murine model were evaluated by choroidal flatmount and fluorescence angiography following formulation and transgenosis of RNA inteference (RNAi) targeting HIF-1α loaded NPs. Additionally, co-culture of retinal pigment epithelial cells (RPE) and choroidal microvascular endothelial cells (CECs) and interaction with BMSCs in high glucose and hypoxia media were studied. This study attemps to offer new thinkings and strategies to prevent and treat CNV related disease under diabetes mellitus. Meanwhile, migration and differentiation of MSCs in vivo and in vitro were investigated by in vivo fluorescent optic imaging, dual whole-cell patch and laser scanning confocal microscope as well as intercellular delivery of siRNA via gap junction, new insight into clinical application of stem cells was provided.

糖尿病是否脉络膜新生血管(CNV)的危险因素尚存争议。本研究组证实，高血糖加重了实验性CNV的发生发展，此时骨髓来源干细胞向CNV局部特异性趋化；缺血缺氧是糖尿病视网膜病变和CNV的共同通路，而针对缺氧诱导因子-1α(HIF-1α)设计小干扰RNA(siRNA)，已显示较好的抑制实验性CNV的作用。然而传统的给药方式影响正常组织的功能，也不能达到缓释给药的目的。本研究拟设计针对HIF-1α的siRNA，合成聚乳酸聚乙醇酸和聚乙烯亚胺共聚物包裹的纳米粒，利用间充质干细胞(MSCs)作为载体，通过实验性CNV模型和细胞共培养模型，观察转染纳米粒的MSCs靶向抑制高糖和低氧刺激CNV的作用，为临床防治糖尿病条件下CNV相关疾病提供新的思路。同时，通过活体荧光成像、膜片钳和激光共聚焦等技术，观察MSCs在体内的移行和分化，以及siRNA通过缝隙连接在细胞间的传递，为干细胞的临床应用提供新思路。

本研究首先在活体动物模型中证实了高血糖可促使更多的骨髓来源细胞(BMC)参与脉络膜新生血管(CNV)的形成，并且CNV程度较正常血糖时加重。因此利用骨髓来源细胞向CNV部位的特异性趋化作用，可以携带抑制药物到CNV部位，并且，在高血糖状态下，这种趋化作用更加明显。在体外建立缺氧和高糖模型，证实载体细胞——间充质干细胞(MSCs)在此微环境中增殖和移行增加，但对凋亡无影响，因此对下一步实验应用建立良好的基础。同时利用视网膜色素上皮细胞(RPE)和MSCs共培养模型，证实缺氧和高糖条件下，效应细胞——RPE细胞增殖和移行增加，不影响凋亡。针对缺氧诱导因子-1α(HIF-1α)设计聚乳酸聚乙醇酸共聚物(PLGA)纳米粒包裹的siRNA，证实其可以抑制RPE细胞中HIF-1α的表达；将PLGA-HIF-1α-siRNA成功搭载MSCs后，证实其不影响MSCs的增殖、移行和凋亡等生物学活性，为下一步的实验应用奠定基础。之后，证实此抑制系统可以降低低氧高糖情况下效应细胞RPE的HIF-1α表达，同时VEGF表达量下降。在体外模型，糖尿病小鼠激光诱导CNV后，通过尾静脉注射搭载了PLGA-HIF-1α-siRNA抑制系统的MSCs细胞，证实此系统抑制了高血糖刺激的CNV发展。.综上所述，在前期研究的基础上，本项目利用了新型缓释系统PLGA-HIF-1α-siRNA搭载MSCs这一新型给药途径，从体内和体外实验证实其可以抑制糖尿病刺激CNV的发生发展，为CNV这一严重影响中心视力和生活质量的疾病治疗提供了新策略。