Exosome-mediated aptamer S58 reduces fibrosis in a rat glaucoma filtration surgery model

INTRODUCTION

In this study, we loaded S58 into exosomes (Exo-S58) and confirmed whether Exo-S58 has a better antifibrotic effect than naked S58 in human conjunctival fibroblast (HConF) cells and in rat GFS models.

Previous reports have demonstrated that transforming growth factor-β (TGF-β) ligands play a crucial role in cell proliferation, migration, conjunctival scarring and wound healing

. As one of the identified TGF-β isoforms, TGF-β

is closely related to the process of conjunctival scarring and fibrosis

. Consequently, TGF-β ligands or receptors are therapeutic targets in the process of postoperative fibrosis through antibody neutralization

, proteoglycan-like decorin inactivation and blockage of exogenous receptors

.Aptamers are single-stranded DNA (ssDNA) or RNA oligonucleotides that can bind to a target protein

and are characterized by low toxicity

, high selectivity and binding affinity

. In our previous study, we applied systematic evolution of ligands by exponential enrichment (SELEX)to identify and synthesize the DNA aptamer S58, which could bind to TGF-β receptor II (TβRII) with high affinity.Experiments

and

proved that aptamer S58 could reduce TGF-β

-induced fibrosis, and fewer myofibroblasts were observed in the S58 group compared with MMC groups

.However, the aptamer’s effective reaction time was limited by nuclease degradation. Thus, it deserves advanced investigation to determine whether a nontoxic and protective delivery vector can preserve the efficacy of aptamer S58.

To observe the change in scarring formation following GFS, the eyeballs from the rats were collected and fixed with 4% paraformaldehyde on postoperative day 14. After dehydration with graded ethanol, the conjunctival tissue was embedded in paraffin. Haematoxylineosin (HE) staining and Masson staining were used to detect the percentage of collagen after 3-μm-thick sections from the eyeballs were sliced. Furthermore, immunofluorescence staining was also performed to determine the tissue fibrosis level. Citrate acid was used to retrieve the antigen from the sections after deparaffination and rehydration. Then, 3%hydrogen peroxide was incubated with the sections in the dark for 25min. After blocking with 1% goat serum albumin for 30min, the sections were incubated with primary antibodies,including mouse anti-α-SMA (ab7817, Abcam) and rabbit anticollagen I (ab260043, Abcam), overnight at 4℃. The sections were incubated with specific secondary antibodies for 1h and stained with DAPI for 5min. Images were captured by a fluorescence microscope (Nikon Eclipse Ti-SR, Nikon, Japan).

All values are presented as the mean±SD.Statistical analysis for comparison of multiple columns or groups was performed by one-way ANOVA using GraphPad Prism 7 (GraphPad, San Diego, CA, USA), and a

＜0.05 was considered statistically significant.

現(xiàn)有文獻(xiàn)鮮有學(xué)者關(guān)注新興旅游部門的自我就業(yè)群體，并衍生研究大學(xué)生創(chuàng)業(yè)可行性。學(xué)者們基本都是從宏觀或中觀的角度分析旅游非正規(guī)就業(yè)及共享經(jīng)濟(jì)下的游客行為，鮮有學(xué)者從微觀的角度去追蹤考察旅游自我就業(yè)者現(xiàn)狀與創(chuàng)業(yè)可行性。最后，隨著失業(yè)問題的靈活就業(yè)彈性方法的提出，由于其主動性、家庭參與性等特征，旅游非正規(guī)就業(yè)在未來可能成為我國旅游地最重要的旅游就業(yè)方式之一，因而對于旅游非正規(guī)就業(yè)研究具有現(xiàn)實意義，所以以番禺沙灣特色小鎮(zhèn)為主的旅游自我就業(yè)現(xiàn)狀與創(chuàng)業(yè)可行性研究是本題研究的首要目標(biāo)。

水結(jié)冰時其體積約增加9%，但水在變成冰的過程中，冰體的壓力并不很大。隨著氣溫的降低，冰的溫度下降，體積變小。當(dāng)溫度再升高時冰則膨脹，如果發(fā)生阻礙冰體膨脹的情況，就產(chǎn)生了作用在鋼閘門上的冰壓力。冰壓力分為靜冰壓力和動冰壓力。靜冰壓力指冰蓋層膨脹對鋼閘門表面產(chǎn)生的冰壓力，靜冰壓力的數(shù)值與冰原、開始升溫時的氣溫及氣溫上升率有關(guān)。動冰壓力是指冰蓋層解凍后，冰塊漂流撞擊鋼閘門時產(chǎn)生的撞擊力。

Glaucoma is recognized as an irreversible progressive neurodegenerative eye disease that can cause optic atrophy, visual field defects and blindness

. According to previous studies, glaucoma is also a multifactorial disease accompanied by pathologically elevated intraocular pressure(IOP) as the most important risk factor

. As such, glaucoma filtration surgery (GFS) has been an integral part of glaucoma control strategies through drainage of the aqueous humour to decrease IOP

. However, surgical failure may occur because of excessive subconjunctival fibrosis and scar formation caused by wound vascular reaction, exudate stimulation and hormones

. Mitomycin C (MMC) and 5-fluorouracil (5-FU)have been generally used to avoid unexpected filtering bleb fibrosis in clinical practice and have side effects such as bleb leakage, endophthalmitis and corneal epithelial toxicity

.Therefore, it is necessary to identify safe and effective antifibrotic alternatives that can be used to prevent scar formation and preserve filtering bleb function after GFS.

MATERIALS AND METHODS

Animal experiments were approved by the Animal Ethics Committee of Chongqing Medical University.All animal procedures complied with the ARVO Statement for the Use of Animals in Ophthalmic and Visual Research.

The human embryonic kidney cell line HEK293T was cultured in complete medium containing 10% exosome-free foetal bovine serum (FBS; Biological Industries, Kibbutz Beit Haemek, Israel), Dulbecco’s modified Eagle’s medium (DMEM; Biological Industries), and 1%cyan-streptomycin (Biological Industries) at 37℃ and 5%CO

. Primary HConFs were cultured in complete medium containing 10% FBS, DMEM and 1% cyan-streptomycin at 37℃ and 5% CO

. In the S58 treatment group, aptamer S58 was first diluted in ddH

O to a final concentration of 50 nmol/L.Exosomes (3.75 μg) loaded with 50 nmol/L S58 were used in the Exo-S58 treatment group. For exosome treatment, 3.75 μg of unloaded exosomes were used. TGF-β

(5 ng/mL, PeproTech,Rocky Hill, NJ, USA) was used for all cell experiments.

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