sgRNA and primer sequences.

BackgroundChoroidal neovascularization (CNV) is a key manifestation of intraocular neovascularization, and it is considered one of the main causes of blindness in ophthalmology. Additionally, multiple anti-vascular endothelial growth factor (VEGF) drugs have been used as first-line treatment for CNV. However, several issues posed challenges to the anti-VEGF drugs, which were mainly composed of short duration of action, requirement for repeated injections, and complications. Thrombospondin-1 (TSP-1) is an endogenous protein that was found to regulate multiple biological processes within the body, and it has been proven to exhibit an inhibitory effect on neovascularization. Besides, the function of TSP-1 during the inhibition of neovascularization was currently considered to mainly focus on its type Ⅰ repeats (TSRs), which was attributed to the large molecular weight, complex structure, and possible unknown functions of TSP-1. Therefore, TSRs can be applied as targets and research directions for the further development and exploration of potential therapeutic drugs.ObjectivesBased on the type I repeats (TSRs) of thrombospondin-1 (TSP-1), amino acid sequences of different lengths were designed and synthesized in this study, named as VR-9 VR-10、VR-11、VR-12、VR-13. The objective was to explore the effects of the above five peptides on angiogenesis in Chori-retinal neovascularization, alongside the screening of the best peptides and the deep exploration into the underlying mechanism, aimed to provide a basis for the development and application of peptide drugs in the treatment of CNV.MethodsWound healing, CCK-8, and 5-ethynyl-2′-deoxyuridine (EdU) assays were employed to evaluate the proliferation and migration ability of cells. CRISPR-Cas9 technology was utilized to establish CD36 knockdown cell lines, alongside the conduction of qPCR to verify the efficiency of gene knockdown. The expression levels of VEGF and CD31 in RF/6A cells and rats were assessed by Western blot. Additionally, Hematoxylin and eosin (HE) staining was performed to examine the structural integrity of the rat retina, while Fluorescein Isothiocyanate-Dextran Cardiac Perfusion (FITC) labeling was used to observe the occurrence and development of choroidal neovascularization (CNV).ResultsAccording to the wound-healing and CCK-8 assays, VR-13 was the most effective in inhibiting the proliferation and migration of endothelial cells. Furthermore, VR-13 peptide effectively inhibited the pathological development of CNV without the detection of retinal toxicity in the rat CNV model.ConclusionsOverall, it was found that VR-13 exhibit significant effects on the inducing of apoptosis and the inhibition of the progression of angiogenesis by regulating the expression of VEGF and CD31 via CD36 signaling pathway.背景：脉络膜新生血管（Choroidal neovascularization，CNV）是眼内新生血管的核心临床表现，被视为眼科主要致盲原因之一。目前已有多种抗血管内皮生长因子（vascular endothelial growth factor，VEGF）药物被用作CNV的一线治疗方案，但此类药物仍存在诸多局限：主要包括作用时长较短、需反复注射以及易引发并发症等问题。血小板反应蛋白-1（Thrombospondin-1，TSP-1）是一种内源性蛋白，可调控体内多种生物学过程，且被证实对新生血管生成具有显著抑制作用。当前研究认为，TSP-1抑制新生血管生成的功能主要与其Ⅰ型重复序列（type Ⅰ repeats，TSRs）相关，这是由于TSP-1本身分子量大、结构复杂且存在潜在未知功能。因此，TSRs可作为潜在治疗药物开发与探索的关键靶点及研究方向。 研究目的：本研究基于血小板反应蛋白-1（TSP-1）的Ⅰ型重复序列（TSRs），设计并合成了不同长度的氨基酸序列，分别命名为VR-9、VR-10、VR-11、VR-12、VR-13。本研究旨在探究上述五种多肽对脉络膜视网膜新生血管生成的影响，筛选出最优多肽，并深入剖析其潜在作用机制，为多肽类药物治疗CNV的开发与临床应用提供理论依据。 研究方法：本研究采用划痕愈合实验、CCK-8实验以及5-乙炔基-2′-脱氧尿苷（5-ethynyl-2′-deoxyuridine，EdU）实验，评估细胞的增殖与迁移能力。利用CRISPR-Cas9技术构建CD36基因敲低细胞系，并通过qPCR验证基因敲低效率。采用Western blot检测RF/6A细胞与大鼠体内VEGF及CD31的表达水平。此外，通过苏木精-伊红（Hematoxylin and eosin，HE）染色观察大鼠视网膜结构完整性，采用异硫氰酸荧光素-葡聚糖心脏灌注（Fluorescein Isothiocyanate-Dextran Cardiac Perfusion，FITC）标记法观察脉络膜新生血管（CNV）的发生与发展进程。 研究结果：划痕愈合实验与CCK-8实验结果显示，VR-13对内皮细胞的增殖与迁移抑制效果最为显著。此外，在大鼠CNV模型中，VR-13多肽可有效阻断CNV的病理进展，且未检测到明显的视网膜毒性。 研究结论：综上，本研究发现VR-13可通过CD36信号通路调控VEGF与CD31的表达水平，进而显著诱导细胞凋亡并抑制新生血管生成进程。