猫角膜内皮中央与周围部端粒酶活性比较及其与增殖潜能关系的研究
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摘要
目的:比较不同年龄猫角膜内皮周围部和中央部细胞的增殖潜能。将神经胶质瘤细胞(U251)与猫角膜内皮细胞(CECs)共培养,观察角膜内皮细胞增殖力及细胞周期的变化,并对共培养后CECs的生物学性能进行初步的评估。
方法:实验对象按年龄不同分为幼年猫组和成年猫组,根据各组猫角膜内皮取材部位不同分为周围部组和中央部组,用实时荧光定量PCR—染料法检测各组角膜内皮端粒酶活性,探讨角膜内皮中央部与周围部增殖潜能是否存在差异。U251细胞、角膜基质细胞分别与猫角膜内皮细胞Transwell体系共培养后倒置相差显微镜观察各组细胞生长情况,通过流式细胞术检测其细胞周期的变化。共培养后的猫角膜内皮细胞进行HE染色观察细胞形态及核分裂情况。并分别收集共培养后的实验组(U251细胞与猫角膜内皮细胞共培养)、阳性对照组(角膜基质细胞与猫角膜内皮细胞共培养)、空白对照组(仅添加培养基的猫角膜内皮细胞)的猫角膜内皮细胞悬液用实时荧光定量PCR法检测端粒酶活性,对各组CECs的生物学性能进行初步的评估。
结果:经实时荧光定量PCR检测猫角膜内皮周围部组和中央部组端粒酶活性表达存在组间差别(F=58.51,P=0.000),周围部组高于中央部组,中央部组仅有微量表达。成年猫组和幼年猫组组间差别无统计学意义(F=6.21,P=0.028),即端粒酶活性表达不受年龄因素影响。U251细胞、角膜基质细胞分别与猫角膜内皮细胞Transwell体系共培养后,流式细胞检测结果实验组G1期比例显著下降较空白组下降10%,S期比例显著提高较空白组提高13%,增殖力明显增强。阳性对照组增殖力略微增强。各组共培养后的猫角膜内皮细胞HE染色观察见实验组细胞数目较对照组明显增多,细胞形态规则,核仁及染色质丰富,偶见大细胞,但未见明显的核异型性,核浆比例正常。共培养后CECs端粒酶活性检测实验组较阳性对照组和空白对照组不同程度增高,但其表达量远低于肿瘤细胞(U251)组。
结论:(1)猫角膜内皮周围部存在“干细胞样细胞”具有增殖潜能,且其增殖能力不受年龄因素影响。(2)与人神经胶质瘤细胞共培养在一定程度上可促进猫角膜内皮细胞增殖且增殖的细胞形态、生物学性能无明显异常改变。
Purpose To compare the proliferative capacity between corneal endothelial cells from the peripheral and central areas of cat corneal.To observe the change of cell proliferative capability and the change of cell cycle after cat corneal endothelial cells(CECs) co-cultured with glioma cells(U251),then make the preliminary evaluation of biological property of co-cultured CECs.
Methods Cat corneas were divided into younger group and older group based on cat's age.We use Real- time fluorescent quantitative PCR to assay the telomerase activity of CECs which were divided into central(0-6.0mm) and peripheral areas(6.0-9.5mm) by the use of trephines.The potential proliferative capacity of CECs both in peripheral and central areas of cornea was investigated.CECs were divided into three groups for the purpose of investigating the factors which promote the CECs' proliferation.CECs were co-cultured with U251 cells in the experimental group and with cat corneal stromal cells in the positive group by transwell systems, and CECs were cultured alone in the control group.The changes of cat corneal endothelial cell cycle were examined by flow cytometry after co-cultured.The morphological changes of cells before and after co-cultured were observed by inverted phase contrast microscope and HE staining,meanwhile,the telomerase activity of CECs were detected by real-time fluorescent quantitative PCR after co-cultured.
Results There is a significant difference of the telomerase activity between the central and peripheral areas(F=58.51,P=0.000) and no statistically significant difference between the younger and older age group(F=6.21,P=0.028) in cats'corneal endothelium.Compared with the control group,both the experimental group and positive group showed the portions of CECs in S-phase increased and G1-phase decreased,which is more obvious in experimental group.The portion of CECs in S-phase of experimental group increased nearly 13%,indicating a significant enhancement of the cell proliferative capacity.After co-cultured,the number of CECs in experimental group has increased,there's no obviously abnormality of CECs morphologically compared with normal CECs.Telomerase activity of CECs in the experimental group increased in different degree compared with those in the positive group and the control group,but it is far less than those of the glioma cells.
Conclusion(1) cat corneal endothelial cells from peripheral areas may be exist stem-like cells and retain a higher potential proliferative capacity which does not related with the age.(2). After co-cultured with U251 cells,the proliferative capacity of CECs shows a significant enhancement,the biological behavior of CECs was not significantly different compare with the normal CECs.
方法:实验对象按年龄不同分为幼年猫组和成年猫组,根据各组猫角膜内皮取材部位不同分为周围部组和中央部组,用实时荧光定量PCR—染料法检测各组角膜内皮端粒酶活性,探讨角膜内皮中央部与周围部增殖潜能是否存在差异。U251细胞、角膜基质细胞分别与猫角膜内皮细胞Transwell体系共培养后倒置相差显微镜观察各组细胞生长情况,通过流式细胞术检测其细胞周期的变化。共培养后的猫角膜内皮细胞进行HE染色观察细胞形态及核分裂情况。并分别收集共培养后的实验组(U251细胞与猫角膜内皮细胞共培养)、阳性对照组(角膜基质细胞与猫角膜内皮细胞共培养)、空白对照组(仅添加培养基的猫角膜内皮细胞)的猫角膜内皮细胞悬液用实时荧光定量PCR法检测端粒酶活性,对各组CECs的生物学性能进行初步的评估。
结果:经实时荧光定量PCR检测猫角膜内皮周围部组和中央部组端粒酶活性表达存在组间差别(F=58.51,P=0.000),周围部组高于中央部组,中央部组仅有微量表达。成年猫组和幼年猫组组间差别无统计学意义(F=6.21,P=0.028),即端粒酶活性表达不受年龄因素影响。U251细胞、角膜基质细胞分别与猫角膜内皮细胞Transwell体系共培养后,流式细胞检测结果实验组G1期比例显著下降较空白组下降10%,S期比例显著提高较空白组提高13%,增殖力明显增强。阳性对照组增殖力略微增强。各组共培养后的猫角膜内皮细胞HE染色观察见实验组细胞数目较对照组明显增多,细胞形态规则,核仁及染色质丰富,偶见大细胞,但未见明显的核异型性,核浆比例正常。共培养后CECs端粒酶活性检测实验组较阳性对照组和空白对照组不同程度增高,但其表达量远低于肿瘤细胞(U251)组。
结论:(1)猫角膜内皮周围部存在“干细胞样细胞”具有增殖潜能,且其增殖能力不受年龄因素影响。(2)与人神经胶质瘤细胞共培养在一定程度上可促进猫角膜内皮细胞增殖且增殖的细胞形态、生物学性能无明显异常改变。
Purpose To compare the proliferative capacity between corneal endothelial cells from the peripheral and central areas of cat corneal.To observe the change of cell proliferative capability and the change of cell cycle after cat corneal endothelial cells(CECs) co-cultured with glioma cells(U251),then make the preliminary evaluation of biological property of co-cultured CECs.
Methods Cat corneas were divided into younger group and older group based on cat's age.We use Real- time fluorescent quantitative PCR to assay the telomerase activity of CECs which were divided into central(0-6.0mm) and peripheral areas(6.0-9.5mm) by the use of trephines.The potential proliferative capacity of CECs both in peripheral and central areas of cornea was investigated.CECs were divided into three groups for the purpose of investigating the factors which promote the CECs' proliferation.CECs were co-cultured with U251 cells in the experimental group and with cat corneal stromal cells in the positive group by transwell systems, and CECs were cultured alone in the control group.The changes of cat corneal endothelial cell cycle were examined by flow cytometry after co-cultured.The morphological changes of cells before and after co-cultured were observed by inverted phase contrast microscope and HE staining,meanwhile,the telomerase activity of CECs were detected by real-time fluorescent quantitative PCR after co-cultured.
Results There is a significant difference of the telomerase activity between the central and peripheral areas(F=58.51,P=0.000) and no statistically significant difference between the younger and older age group(F=6.21,P=0.028) in cats'corneal endothelium.Compared with the control group,both the experimental group and positive group showed the portions of CECs in S-phase increased and G1-phase decreased,which is more obvious in experimental group.The portion of CECs in S-phase of experimental group increased nearly 13%,indicating a significant enhancement of the cell proliferative capacity.After co-cultured,the number of CECs in experimental group has increased,there's no obviously abnormality of CECs morphologically compared with normal CECs.Telomerase activity of CECs in the experimental group increased in different degree compared with those in the positive group and the control group,but it is far less than those of the glioma cells.
Conclusion(1) cat corneal endothelial cells from peripheral areas may be exist stem-like cells and retain a higher potential proliferative capacity which does not related with the age.(2). After co-cultured with U251 cells,the proliferative capacity of CECs shows a significant enhancement,the biological behavior of CECs was not significantly different compare with the normal CECs.
引文
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[2]周道伐 朱志忠 角膜内皮细胞的研究进展[J]国际眼科学纵览 1980年05期
[3]Joyce NC,Harris DL,Mello DM.Mechanisms of mitotic inhibition in corneal endothelium:contact inhibition and TGF-beta2[J].Invest Ophthalmol Vis Sci,2002;43:2152-2159.
[4]Joyce NC.Proliferative capacity of the corneal endothelium[J].Prog Retin Eye Res,2003,22:35923891
[5]Joyce NC,Harris DL,Mello DM.Mechanisms of mitotic inhibition in corneal endothelium:contact inhibition and TGF-beta2[J].Invest Ophthalmol Vis Sci 2002;43:2152-9.
[6]Bourne WM,Nelson LR,Hodge DO.Central corneal endothelial cell changes over a ten-year period[J].Invest Ophthalmol Vis Sci1997;38:779-82
[7]Hoppenreijs VP,Pels E,Vrensen GF,.Ef-fects of human epidermal growth factor on endothelial wound healing of human corneas[J].Invest Ophthalmol Vis Sci 1992;33:1946-57.33:1946-57.
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