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ORIGINAL ARTICLE
Year : 2018  |  Volume : 131  |  Issue : 6  |  Page : 704-712

Effects of Adipose-derived Mesenchymal Stem Cell Exosomes on Corneal Stromal Fibroblast Viability and Extracellular Matrix Synthesis


1 Eye Institute of Zhejiang University, Eye Center of Second Affiliated Hospital of Zhejiang University, School of Medicine, Hangzhou, Zhejiang 310009; Department of Ophthalmology, Zhejiang Provincial People's Hospital and People's Hospital of Hangzhou Medical College, Hangzhou, Zhejiang 310014, China
2 Department of Ophthalmology, Zhejiang Provincial People's Hospital and People's Hospital of Hangzhou Medical College, Hangzhou, Zhejiang 310014, China
3 School of Ophthalmology and Optometry, Wenzhou Medical University, Wenzhou, Zhejiang 325035, China
4 Facility for Biochemistry and Molecular Medicine Core Facilities, School of Medicine, Zhejiang University, Hangzhou, Zhejiang 310058, China
5 Department of Ophthalmology, Zhejiang Provincial People's Hospital and People's Hospital of Hangzhou Medical College, Hangzhou, Zhejiang 310014, ; School of Ophthalmology and Optometry, Wenzhou Medical University, Wenzhou, Zhejiang 325035, China
6 Eye Institute of Zhejiang University, Eye Center of Second Affiliated Hospital of Zhejiang University, School of Medicine, Hangzhou, Zhejiang 310009, China

Correspondence Address:
Prof. Ke Yao
Eye Institute of Zhejiang University, Eye Center of Second Affiliated Hospital of Zhejiang University, School of Medicine, Hangzhou, Zhejiang 310009
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0366-6999.226889

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Background: Corneal stromal cells (CSCs) are components of the corneal endothelial microenvironment that can be induced to form a functional tissue-engineered corneal endothelium. Adipose-derived mesenchymal stem cells (ADSCs) have been reported as an important component of regenerative medicine and cell therapy for corneal stromal damage. We have demonstrated that the treatment with ADSCs leads to phenotypic changes in CSCs in vitro. However, the underlying mechanisms of such ADSC-induced changes in CSCs remain unclear. Methods: ADSCs and CSCs were isolated from New Zealand white rabbits and cultured in vitro. An Exosome Isolation Kit, Western blotting, and nanoparticle tracking analysis (NTA) were used to isolate and confirm the exosomes from ADSC culture medium. Meanwhile, the optimal exosome concentration and treatment time were selected. Cell Counting Kit-8 and annexin V-fluorescein isothiocyanate/propidium iodide assays were used to assess the effect of ADSC- derived exosomes on the proliferation and apoptosis of CSCs. To evaluate the effects of ADSC- derived exosomes on CSC invasion activity, Western blotting was used to detect the expression of matrix metalloproteinases (MMPs) and collagens. Results: ADSCs and CSCs were successfully isolated from New Zealand rabbits. The optimal concentration and treatment time of exosomes for the following study were 100 μg/ml and 96 h, respectively. NTA revealed that the ADSC-derived exosomes appeared as nanoparticles (40–200 nm), and Western blotting confirmed positive expression of CD9, CD81, flotillin-1, and HSP70 versus ADSC cytoplasmic proteins (all P < 0.01). ADSC-derived exosomes (50 μg/ml and 100 μg/ml) significantly promoted proliferation and inhibited apoptosis (mainly early apoptosis) of CSCs versus non-exosome-treated CSCs (all P < 0.05). Interestingly, MMPs were downregulated and extracellular matrix (ECM)-related proteins including collagens and fibronectin were upregulated in the exosome-treated CSCs versus non-exosome-treated CSCs (MMP1: t = 80.103, P < 0.01; MMP2: t = 114.778, P < 0.01; MMP3: t = 56.208, P < 0.01; and MMP9: t = 60.617, P < 0.01; collagen I: t = −82.742, P < 0.01; collagen II: t = −72.818, P < 0.01; collagen III: t = −104.452, P < 0.01; collagen IV: t = −133.426, P < 0.01, and collagen V: t = −294.019, P < 0.01; and fibronectin: t = −92.491, P < 0.01, respectively). Conclusion: The findings indicate that ADSCs might play an important role in CSC viability regulation and ECM remodeling, partially through the secretion of exosomes.

 

 Abstract in Chinese

脂肪来源间质干细胞外泌体对角膜基质纤维成纤维细胞活性和胞外基质合成的影响

摘要

背景:角膜基质细胞(CSCs)是角膜内皮微环境的重要组分,可诱导构建功能性组织工程角膜。脂肪来源干细胞(ADSCs)被认为是角膜基质损伤的再生医学细胞治疗的重要方法之一。我们已经发现ADSCs在体外能诱导CSCs表型的改变,但其机制仍不明确。

方法:ADSCs与CSCc分离自新西兰大白兔并在体外培养。使用外泌体提取试剂盒、蛋白免疫印迹法、纳米颗粒跟踪分析技术提取验证脂肪干细胞培养基中的外泌体,同时确定最佳的外泌体浓度与处理时间。使用细胞增殖检测试剂盒和流式双染色法分析脂肪干细胞外泌体对角膜基质细胞增殖和凋亡的影响。使用细胞免疫印迹法检测基质金属蛋白酶和胶原的表达,评估脂肪干细胞外泌体对角膜基质细胞迁徙活性的作用。

结果:成功分离ADSCs与CSCs,发现最佳外泌体实验浓度为100μg/ml,对角膜基质细胞处理最佳时间为96小时。脂肪干细胞外泌体为直径在40-200nm纳米颗粒,与ADSC胞质蛋白相比,CD9、CD81、脂阀结构蛋白1和 HSP70表达阳性(P <0.01)。50 μg/ml和100 μg/ml浓度的外泌体处理CSCs与单纯CSCs培养相比,外泌体显著促进CSCs增殖、抑制凋亡(主要为早期凋亡;P均<0.05)。在外泌体处理CSCs组中,基质金属蛋白酶(MMPs)下调,包括MMP1(t=80.103, P<0.01)、MMP2(t=114.778, P<0.01)、MMP3(t=56.208, P<0.01)、MMP9(t=60.617, P<0.01),细胞外基质蛋白增多,包括I型(t=-82.742, P<0.01)、II型(t=-72.818, P<0.01)、III型(t=-104.452, P<0.01)、IV型(t=-133.426, P<0.01)、V型胶原(t=-294.019, P<0.01)、纤维连接蛋白(t=-92.491, P<0.01)。

结论:本研究发现提示ADSCs在CSCs活性调控和胞外基质重塑中有重要作用,与其分泌外泌体有一定相关。



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