中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2014年
1期
28-31
,共4页
辛晓蓉%巩天祥%赵莉%李新章
辛曉蓉%鞏天祥%趙莉%李新章
신효용%공천상%조리%리신장
缺氧%脑膜上皮细胞%线粒体%细胞色素C%脑脊液-视神经屏障
缺氧%腦膜上皮細胞%線粒體%細胞色素C%腦脊液-視神經屏障
결양%뇌막상피세포%선립체%세포색소C%뇌척액-시신경병장
Hypoxia%Meningothelial cells%Mitochondria%Cytochrome C%Cerebral spinal fluid-optic nerve barrier
背景 脑膜上皮细胞(MECs)是构成脑脊液-视神经屏障的主要细胞成分.脑脊液-视神经屏障的损害可能会导致脑脊液组分的失衡,使视神经受到各种致病因素的攻击.目前对于MECs在视神经疾病中的病理作用研究甚少,机制尚不明确. 目的 探讨缺氧条件下人MECs的功能变化,为视神经疾病发病机制的研究提供新的线索.方法 将培养的人MECs株分别制备成密度为2.5×103个/孔、5.0×103个/孔和1×104个/孔的细胞悬液,各取100μl分别接种于96孔板中,分别在常规培养基及体积分数21％O2(常氧组)和1％O2(缺氧组)环境下孵育2d,采用MTS法测定和比较不同氧环境下人MECs的吸光度(A490)值;采用CASY1法检测和比较不同氧环境下细胞体积及直径的变化;采用光度计测定MECs暴露不同氧环境下1d、2d后线粒体产生ATP量的变化;采用免疫荧光技术测定不同氧环境下细胞内细胞色素C的表达和定位. 结果 缺氧组2.5× 103个/孔、5.0×103个/孔、1×104个/孔细胞密度组MECs的增生值(A490)分别为0.399±0.009、0.393±0.009和0.496±0.026,分别较相应的常氧组的0.424±0.131、0.413±0.111和0.537±0.021明显下降,差异均有统计学意义(t=3.777,P=O.004;t=3.251,P=O.009;t=3.037,P=O.013).与常氧组细胞比较,缺氧组细胞的直径和体积均明显增加[(20.970±0.127) μm vs.(21.198±0.048) μm,t=-3.762,P=0.006;(5805±73)fl vs.(6026±106) fl,t=-4.124,P=O.002)].缺氧组和缺氧+底物组细胞分别培养2d后,细胞中ATP产生量分别为(0.900±0.225) mmol/(L·g)、(0.952±0.075) mmol/(L·g),均明显低于常氧组的(1.389±0.145)mmol/(L·g)和常氧+底物组的(1.401±0.122)mmol/(L·g),差异均有统计学意义(P=O.001、0.002、0.001).常氧组细胞中细胞色素C的绿色荧光主要分布于线粒体,而缺氧组MECs中细胞色素C的释放弥散分布于细胞质. 结论 缺氧环境下MECs的生理功能明显减退,推测MECs的功能损害是脑脊液和视神经之间的屏障完整性受到损害的主要机制.
揹景 腦膜上皮細胞(MECs)是構成腦脊液-視神經屏障的主要細胞成分.腦脊液-視神經屏障的損害可能會導緻腦脊液組分的失衡,使視神經受到各種緻病因素的攻擊.目前對于MECs在視神經疾病中的病理作用研究甚少,機製尚不明確. 目的 探討缺氧條件下人MECs的功能變化,為視神經疾病髮病機製的研究提供新的線索.方法 將培養的人MECs株分彆製備成密度為2.5×103箇/孔、5.0×103箇/孔和1×104箇/孔的細胞懸液,各取100μl分彆接種于96孔闆中,分彆在常規培養基及體積分數21％O2(常氧組)和1％O2(缺氧組)環境下孵育2d,採用MTS法測定和比較不同氧環境下人MECs的吸光度(A490)值;採用CASY1法檢測和比較不同氧環境下細胞體積及直徑的變化;採用光度計測定MECs暴露不同氧環境下1d、2d後線粒體產生ATP量的變化;採用免疫熒光技術測定不同氧環境下細胞內細胞色素C的錶達和定位. 結果 缺氧組2.5× 103箇/孔、5.0×103箇/孔、1×104箇/孔細胞密度組MECs的增生值(A490)分彆為0.399±0.009、0.393±0.009和0.496±0.026,分彆較相應的常氧組的0.424±0.131、0.413±0.111和0.537±0.021明顯下降,差異均有統計學意義(t=3.777,P=O.004;t=3.251,P=O.009;t=3.037,P=O.013).與常氧組細胞比較,缺氧組細胞的直徑和體積均明顯增加[(20.970±0.127) μm vs.(21.198±0.048) μm,t=-3.762,P=0.006;(5805±73)fl vs.(6026±106) fl,t=-4.124,P=O.002)].缺氧組和缺氧+底物組細胞分彆培養2d後,細胞中ATP產生量分彆為(0.900±0.225) mmol/(L·g)、(0.952±0.075) mmol/(L·g),均明顯低于常氧組的(1.389±0.145)mmol/(L·g)和常氧+底物組的(1.401±0.122)mmol/(L·g),差異均有統計學意義(P=O.001、0.002、0.001).常氧組細胞中細胞色素C的綠色熒光主要分佈于線粒體,而缺氧組MECs中細胞色素C的釋放瀰散分佈于細胞質. 結論 缺氧環境下MECs的生理功能明顯減退,推測MECs的功能損害是腦脊液和視神經之間的屏障完整性受到損害的主要機製.
배경 뇌막상피세포(MECs)시구성뇌척액-시신경병장적주요세포성분.뇌척액-시신경병장적손해가능회도치뇌척액조분적실형,사시신경수도각충치병인소적공격.목전대우MECs재시신경질병중적병리작용연구심소,궤제상불명학. 목적 탐토결양조건하인MECs적공능변화,위시신경질병발병궤제적연구제공신적선색.방법 장배양적인MECs주분별제비성밀도위2.5×103개/공、5.0×103개/공화1×104개/공적세포현액,각취100μl분별접충우96공판중,분별재상규배양기급체적분수21％O2(상양조)화1％O2(결양조)배경하부육2d,채용MTS법측정화비교불동양배경하인MECs적흡광도(A490)치;채용CASY1법검측화비교불동양배경하세포체적급직경적변화;채용광도계측정MECs폭로불동양배경하1d、2d후선립체산생ATP량적변화;채용면역형광기술측정불동양배경하세포내세포색소C적표체화정위. 결과 결양조2.5× 103개/공、5.0×103개/공、1×104개/공세포밀도조MECs적증생치(A490)분별위0.399±0.009、0.393±0.009화0.496±0.026,분별교상응적상양조적0.424±0.131、0.413±0.111화0.537±0.021명현하강,차이균유통계학의의(t=3.777,P=O.004;t=3.251,P=O.009;t=3.037,P=O.013).여상양조세포비교,결양조세포적직경화체적균명현증가[(20.970±0.127) μm vs.(21.198±0.048) μm,t=-3.762,P=0.006;(5805±73)fl vs.(6026±106) fl,t=-4.124,P=O.002)].결양조화결양+저물조세포분별배양2d후,세포중ATP산생량분별위(0.900±0.225) mmol/(L·g)、(0.952±0.075) mmol/(L·g),균명현저우상양조적(1.389±0.145)mmol/(L·g)화상양+저물조적(1.401±0.122)mmol/(L·g),차이균유통계학의의(P=O.001、0.002、0.001).상양조세포중세포색소C적록색형광주요분포우선립체,이결양조MECs중세포색소C적석방미산분포우세포질. 결론 결양배경하MECs적생리공능명현감퇴,추측MECs적공능손해시뇌척액화시신경지간적병장완정성수도손해적주요궤제.
Background BackgroundMeningothelial cells (MECs) are major cell type in the meningeal sheath around optic nerve,which form a fluid barrier between optic nerve and the cerebral spinal fluid.The impairment of the cerebral fluid-optic nerve barrier probably affects the balance of cerebral fluid components.Currently,the investigation on the role of MECs in neuropathy is less performed.Objective This study attempted to explore hypoxia-induced function changes of MECs,and to shed a new clus for the future research of optic nerve disorders.Methods Human MECs strains were cultured in vitro and cell suspension was prepared with the cell densities of 2.5 ×103/hole,5.0× 103/hole and 1 x 104 /hole,respectively.The suspensions of 100 μl were separately collected to incubate in 96-well plates and cultivated for 2 days in 21％ O2(normoxia group) or 1％ O2(hypoxia group).MTS was used to detect and compare the proliferative value (A490) of MECs between the normoxia group and the hypoxia group.The changes of MECs diameter and volume were measured by CASY1 assay.ATP product in the cells after MECs exposed to different oxygen environments with or without substrate (100 mmol/L pyruvate and 100 mmol/L malate) for 1,2 days were assayed by Luminometer method.The expression and distribution of cytochrome C in the cells of the normoxia group and the hypoxia group were determined by immunofluorescence.Results A490 of MECs in the 2.5× 103/hole,5.0× 103/hole and 1 × 104/hole were 0.399±0.009,0.393±0.009 and 0.496±0.026 in the hypoxia group,which were lower than 0.424±0.131,0.413±0.111 and 0.537±0.021 in the normoxia group (t =3.777,P =0.004 ; t =3.251,P =0.009 ; t =3.037,P =0.013).Compared with the normoxia group,the diameter and volume were significantly increased in the hypoxia group ([20.970 ±0.127] μm vs.[21.198 ±0.048] μm,t =-3.762,P=0.006; [5805±73] fl vs.[6026±106] fl,t=-4.124,P=0.002).ATP products were (0.900±0.225)mmol/(L· g) and (0.952± 0.075) mmol/(L · g) in the hypoxia group and the hypoxia+substrate group,which were significantly lower than (1.389±0.145) mmol/(L · g) and (1.401±0.122) mmol/(L · g) in the normoxia group and the normoxia +substrate group (P =0.001,0.002,0.001).Immunofluorescense staining showed that the green fluorescence of cytochrome C located at mitochondria of MECs in the normoxia group,but in the hypoxia group,cytochrome C distributed in the cytoplasm extensively.Conclusions Hypoxia induces malfunction of MECs,which might impact the intact of the cerebral spinal fluid-optic nerve barrier and therefore influence the microenvironment of the subarachnoid space and neuronal function.
