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Cell viability and extracellular matrix synthesis in a co-culture system of corneal stromal cells and adipose-derived mesenchymal stem cells

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International Journal of Ophthalmology(English edition) 2017年第5期10卷 670-678页

作者： Ting Shen Jiang Shen Qing-Qing Zheng Qiu-Shi Li Hai-Lan Zhao Lei Cui Chao-Yang Hong Zhejiang Provincial People's Hospital Hangzhou 310014Zhejiang Province China Beijing Shijitan Hospital Beijing 100050 China Wenzhou Medical University Wenzhou 325035 Zhejiang Province China

AIM：To investigate the impact of adipose-derived mesenchymal stem cells（ADSCs） on cell viability and extracellular matrix（ECM） synthesis of corneal stromal cells（CSCs）. METHODS：ADSCs and CSCs were obtained from the corneas of New Zealand white rabbits and indirectly cocultured in vitro. The proliferative capacity of CSCs in the different groups was assessed by CCK-8 assays. Annexin V-fluorescein isothiocyanate（FITC）/proliferation indices（PI） assays were used to detect the apoptosis of CSCs. The expression levels of matrix metalloproteinase（MMP）, such as MMP1, MMP2, MMP9, and collagens were also evaluated by Western blot. RESULTS：ADSCs significantly promoted proliferation and invasion of CSCs in the indirect co-culture assays. The co-cultural group displayed much higher ability of proliferation, especially under the co-culture conditions of ADSCs for 3d, compared with that CSCs cultured alone. The PI of CSCs in the co-culture system were increased approximately 3-8-fold compared with the control group. A significant change was observed in the proportions of cells at apoptosis（early and late） between the negative control group（6.34% and 2.06%） and the ADCSs-treated group（4.69% and 1.59%）. The expression levels of MMPs were down regulated in the co-culture models. Compared with the control group, the decrease intensities of MMP-1, MMP-2 and MMP-9 in CSCs/ADSCs group were observed, 3.90-fold, 1.09-fold and 3.03-fold, respectively. However, the increase intensities of collagen type（I, II, III, IV, and V） in CSCs were observed in CSCs/ADSCs group, 3.47-fold,4.30-fold, 2.35-fold, 2.55-fold and 2.43-fold, respectively, compared to that in the control group. The expressions of aldehyde dehydrogenase and fibronectin in CSCs were upregulated in the co-culture ***：ADSCs play a promotive role in CSCs＇ growth and invasion, which may be partially associated with MMPs decrease and collagens increase, resulting in a positive participation in the plasticity and ECM synt

关键词： adipose-derived mesenchymal stem cell corneal stromal cells extracellular matrix plasticity

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corneal stromal cells restoration in vitro by exosome derived from ADSCs

Corneal stromal cells restoration in vitro by exosome derive...

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2018年浙江省医学会激光医学分会学术年会暨浙江省医学会激光医学分会第二届学术年会

作者： Ting Shen Qingqing Zheng Jiang Shen Zhejiang Provincial people’s Hospital

Adipose-derived mesenchymal stem cells(ADSCs) have been reported as an important component of the regenerative *** stromal cells(CSCs),as components of the corneal endothelial microenvironment,can be induced into a functional tissue-engineered corneal *** have demonstrated that ADSCs treatment leads to the phenotypic changes of CSCs in ***,the underlying mechanisms remain ***,we evaluated the effects of ADSCs-derived exosomes on CSCs in vitro using CCK-8 assays,Annexin V-FITC/PI assays,western blot and morphological *** tracking analysis technique(NTA) revealed ADSCsderived exosomes appeared as nanoparticles(40-200 nm),and western blot confirmed its positive expression of CD9,CD81,flotillin-1,and *** ADSCs exosome-treated CSCs showed significant difference in proliferation,apoptosis,especially the expression of extracellular matrix(ECM) related proteins,compared to the control ***-derived exosomes significantly promoted proliferation and inhibited the apoptosis of ***,MMPs were downregulated and collagens were upregulated in exosomes-treated *** of MMPs partially impeded the degradation of ECM,and increase of collagens contributed the ECM synthesis in *** findings indicate that ADSCs play an important role during CSCs’ ECM remodeling,which is closely related to phenotypic changes,partially through the secretion of exosomes.

关键词： Adipose-derived mesenchymal stem cell corneal stromal cells extracellular matrix synthesis exosomes

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

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Chinese Medical Journal 2018年第6期131卷 704-712页

作者： Ting Shen Qing-Qing Zheng Jiang Shen Qiu-Shi Li Xing-Hui Song Hong-Bo Luo Chao-Yang Hong Ke Yao Eye Institute of Zhejiang University Eye Center of Second Affiliated Hospital of Zhejiang University School of Medicine Hangzhou Zhejiang 310009 China Department of Ophthalmology Zhejiang Provincial People's Hospital and People's Hospital of Hangzhou Medical College Hangzhou Zhejiang 310014 China School of Ophthalmology and Optometry Wenzhou Medical University Wenzhou Zhejiang 325035 China Facility for Biochemistry and Molecular Medicine Core Facilities School of Medicine Zhejiang University Hangzhou Zhejiang 310058 China

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 ofADSC-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, CDSI, flotillin-1, and HSP70 versus ADSC cytoplasmic proteins （all P〈 0.01 ）. ADSC-derived exosomes （50μg/ml and 100μg/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-

关键词： Adipose-derived Mesenchymal Stem Cell corneal stromal cells Exosomes Extracellular Matrix Synthesis

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