骨髓来源细胞表达的MMP-2/MMP-13在脉络膜新生血管发生发展中的作用
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摘要
目的探讨骨髓来源细胞(BMCs)基质金属蛋白酶(MMP)的动态表达及其与脉络膜新生血管(CNV)发生、发展的关系。方法选取成功接受骨髓移植的24只C57BL/6J绿色荧光蛋白(GFP)嵌合体小鼠为观察组,未接受骨髓移植的48只C57BL/6J小鼠为对照组。两组均予激光光凝照射以诱发CNV,利用Western blot法及明胶酶谱法检测对照组在激光诱发前、诱发后1、3、5、7、10、14、28 d时MMP-2/MMP-13的表达情况,利用免疫荧光染色检测观察组CNV中GFP阳性细胞(即移植的骨髓细胞)是否表达MMP-2/MMP-13,并进行定量分析。结果 Western blot法、明胶酶谱法、免疫荧光均显示在激光诱发早期两组CNV中MMP-2的表达即开始快速增加,在诱导第3天表达量最高、活性最强,且观察组BMCs表达MMP-2也在此时达到高峰,占总表达量的63.21%,随后MMP-2表达量开始下降;在激光诱发早期两组CNV中MMP-13的表达缓慢增加,在诱导后第7天表达量最高、活性最强,且观察组BMCs表达MMP-13亦达最高峰,占总表达量的77.87%,随后下降。结论 BMCs可能作为调控靶点改变MMP-2/MMP-13的表达,进而影响血管细胞的出芽、移行、凋亡及CNV纤维化。
Objective To investigate matrix metalloproteinase( MMP) dynamic expression by bone marrow-derived cells( BMCs) and its relationship with pathogenesy and development of choroidal neovascularization( CNV). Methods A total of 24 C57BL/6J green fluorescent protein( GFP) of allophenic mice,who underwent bone marrow transplantation successfully,were selected as observation group,while other 48 C57BL/6J mice without bone marrow transplantation were selected as control group. CNV was induced by irradiating with laser photocoagulation for all mice. In control group,MMP-2/MMP-13 expressions were detected by Western blot and gelatin zymography method before induction,and at 1st,3rd,5th,7th,10 th,14thand 28 thd after induction; In observation group,immunofluorescence staining was used to identify whether or not MMP-2/MMP-13 was expressed by GFP-positive BMCs in CNV,and quantitative analysis was also performed. Results Western blot,gelatin zymography and immunofluorescence staining methods all showed that MMP-2 expressions were rapidly increased in the early laser stage of CNV patients in two groups,and the expressions of BMCs and MMP-2 reached the highest values and strongest activity at 3rdd after induction; the highest value of MMP-2 expression in observation group occupied 63. 21% of the total expression; and then MMP-2 expression began to decline. MMP-13 expression showed slowly increases at the early laser stage of CNV patients in two groups,and the highest expression and strongest activity were found at 7thd after induction; the highest values of MMP-13 expression in CNV patients in two groups and in observation group occupied 77. 87% of the total expression; and then MMP-13 expression began to decline. Conclusion BMCs can be used as target point to regulate MMP-2/MMP-13 expression,and it can further affect pullulation,transmigration,apoptosis and fibration of CNV.
Objective To investigate matrix metalloproteinase( MMP) dynamic expression by bone marrow-derived cells( BMCs) and its relationship with pathogenesy and development of choroidal neovascularization( CNV). Methods A total of 24 C57BL/6J green fluorescent protein( GFP) of allophenic mice,who underwent bone marrow transplantation successfully,were selected as observation group,while other 48 C57BL/6J mice without bone marrow transplantation were selected as control group. CNV was induced by irradiating with laser photocoagulation for all mice. In control group,MMP-2/MMP-13 expressions were detected by Western blot and gelatin zymography method before induction,and at 1st,3rd,5th,7th,10 th,14thand 28 thd after induction; In observation group,immunofluorescence staining was used to identify whether or not MMP-2/MMP-13 was expressed by GFP-positive BMCs in CNV,and quantitative analysis was also performed. Results Western blot,gelatin zymography and immunofluorescence staining methods all showed that MMP-2 expressions were rapidly increased in the early laser stage of CNV patients in two groups,and the expressions of BMCs and MMP-2 reached the highest values and strongest activity at 3rdd after induction; the highest value of MMP-2 expression in observation group occupied 63. 21% of the total expression; and then MMP-2 expression began to decline. MMP-13 expression showed slowly increases at the early laser stage of CNV patients in two groups,and the highest expression and strongest activity were found at 7thd after induction; the highest values of MMP-13 expression in CNV patients in two groups and in observation group occupied 77. 87% of the total expression; and then MMP-13 expression began to decline. Conclusion BMCs can be used as target point to regulate MMP-2/MMP-13 expression,and it can further affect pullulation,transmigration,apoptosis and fibration of CNV.
引文
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[14]高凡,王雨生,侯慧媛.两种缺氧模型对小鼠骨髓间充质干细胞中基质金属蛋白酶表达及其对促血管形成能力的影响[J].眼科新进展,2016,36(5):401-405.
[15]Gao F,Sun M,Gong Y,et al.Micro RNA-195a-3p inhibits angiogenesis by targeting Mmp2 in murine mesenchymal stem cells[J].Mol Reprod Dev,2016,83(5):413-423.
[2]Hoffmann S,He S,Ehren M,et al.MMP-2 and MMP-9secretion by rpe is stimulated by angiogenic molecules found in choroidal neovascular membranes[J].Retina,2006,26(4):454-461.
[3]Barnett J M,Penn J S,Jayagopal A.Imaging of endothelial progenitor cell subpopulations in angiogenesis using quantum dot nanocrystals[J].Methods Mol Biol,2013,1026:45-56.
[4]Hou H Y,Liang H L,Wang Y S.Bone marrow-derived cells in neovascular age-related macular degeneration:contribution and potential application[J].Ophthalmic Res,2011,45(1):1-4.
[5]Hou H Y,Wang Y S,Xu J F,et al.The dynamic conduct of bone marrow-derived cells in the choroidal neovascularization microenviroment[J].Curr Eye Res,2006,31(12):1051-1061.
[6]Hou H Y,Wang Y S,Xu J F,et al.Nicotine promotes contribution of bone marrow-derived cells to experimental choroidal neovascularization in mice[J].Exp Eye Res,2008,86(6):983-990.
[7]Lecomte J,Louis K,Detry B,et al.Bone marrow-derived mesenchymal cells and MMP13 contribute to experimental choroidal neovascularization[J].Cell Mol Life Sci,2011,68(4):677-686.
[8]Espinosa-Heidmann D G,Caicedo A,Hernandez E P,et al.Bone marrow-derived progenitor cells contribute to experimental choroidal neovascularization[J].Invest Ophthalmol Vis Sci,2003,44(11):4914-4919.
[9]杨秀梅,王雨生.脉络膜新生血管的动物模型[J].国际眼科纵览,2006,30(3):166-170.
[10]侯慧媛,王雨生,徐建峰.骨髓来源细胞在脉络膜新生血管生成中的作用[J].中华眼底病杂志,2009,25(1):30-33.
[11]Tong J P,Yao Y F.Contribution of VEGF and PEDF to choroidal angiogenesis:a need for balanced expressions[J].Clin Biochem,2006,39(3):267-276.
[12]Chung C,Shugrue C,Nagar A,et al.Ethanol exposure depletes hepatic pigment epithelium-derived factor,a novel lipid regulator[J].Gastroenterology,2009,136(1):331-340.
[13]Hou H Y,Liang H L,Wang Y S,et al.A therapeutic strategy for choroidal neovascularization based on recruitment of mesenchymal stem cells to the sites of lesions[J].Mol Ther,2010,18(10):1837-1845.
[14]高凡,王雨生,侯慧媛.两种缺氧模型对小鼠骨髓间充质干细胞中基质金属蛋白酶表达及其对促血管形成能力的影响[J].眼科新进展,2016,36(5):401-405.
[15]Gao F,Sun M,Gong Y,et al.Micro RNA-195a-3p inhibits angiogenesis by targeting Mmp2 in murine mesenchymal stem cells[J].Mol Reprod Dev,2016,83(5):413-423.