中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2013年
5期
446-451
,共6页
夏静%张晓峰%夏蔚%钟蕾%孙正太%王英明
夏靜%張曉峰%夏蔚%鐘蕾%孫正太%王英明
하정%장효봉%하위%종뢰%손정태%왕영명
单色光%Müller细胞%近视%转化生长因子-β1%酪氨酸羟化酶%诱导型一氧化氮合酶%碱性成纤维细胞生长因子
單色光%Müller細胞%近視%轉化生長因子-β1%酪氨痠羥化酶%誘導型一氧化氮閤酶%堿性成纖維細胞生長因子
단색광%Müller세포%근시%전화생장인자-β1%락안산간화매%유도형일양화담합매%감성성섬유세포생장인자
Monochromatic light%Müller cell%Myopia%Transforming growth factor-beta 1%Tyrosine hydroxylase%Inducible nitric oxide synthase%Basic fibroblast growth factor
背景 530 nm单色光照射可诱导动物产生近视.Müller细胞作为视网膜上主要的胶质细胞,参与近视的形成,但Müller细胞在单色光诱导的近视形成过程中的具体作用尚不清楚. 目的 研究530 nm单色光照射对体外培养的大鼠视网膜Müller细胞的生物学特征及近视相关细胞因子表达的影响,探讨Müller细胞在单色光诱导的近视形成中的作用.方法 在自制光照度分别为125、250、500 lx波长530 nm单色光设备的细胞培养箱中用常规细胞培养基培养永生化大鼠Müller细胞,另用无光照的常规培养法培养细胞作为对照组.细胞培养6、12、24 h后,用四甲基偶氮噻唑蓝(MTT)法检测细胞在波长570 nm处的吸光度(A570)值；培养48 h后用流式细胞仪检测各组大鼠Müller细胞的细胞周期,比较各组G2期与G1期细胞数的比值；采用逆转录PCR(RT-PCR)法检测各组大鼠Müller细胞中转化生长因子-β1(TGF-β1)、酪氨酸羟化酶(TH)、诱导型一氧化氮合成酶(iNOS)和碱性成纤维细胞生长因子(bFGF) mRNA表达量的改变.采用两因素方差分析比较各组间大鼠Müller细胞随不同培养时间的变化增生值的差异,采用单因素方差分析比较各组间不同细胞周期细胞数的变化以及大鼠Müller细胞中各近视相关因子mRNA表达量的差异. 结果 530 nm单色光照射后,Müller细胞均呈典型的多角形,细胞大小均匀,边界清晰.与对照组比较,125 lx组、250 lx组大鼠Müller细胞在培养0、6、12、24 h时A570值的差异均无统计学意义(P＞0.05),但500 lx组光照12h、24 h后细胞A570值明显低于对照组,差异均有统计学意义(P=0.013、0.001).与对照组比较,125 lx组、250 lx组大鼠Müller细胞G2/G1细胞数比值的差异均无统计学意义(P=0.073、0.330),500 lx组G2/G1细胞数比值明显降低,与对照组、250 lx组比较差异均有统计学意义(P=0.028、0.038).RT-PCR检测结果显示,250 lx组TGF-β1 mRNA的表达明显高于500 lx组(P=0.006)；125 lx组、250 lx组iNOS mRNA表达依次上调,250 lx组与对照组比较差异有统计学意义(P=0.001),而500 lx组表达下调,与对照组比较差异有统计学意义(P=0.000).与对照组比较,125 lx组、250 lx组bFGF mRNA的表达量均上调,但500 lx组细胞中bFGF mRNA表达量下调,差异均有统计学意义(P=0.002、0.000、0.005).与对照组比较,250 lx组细胞中TH mRNA的表达量明显增加,500 lx组细胞中TH mRNA表达量明显下调,差异均有统计学意义(P=0.000、0.001). 结论 光照度＞250 lx的530 nm单色光照射可以通过抑制大鼠Müller细胞的生长,并下调细胞中近视相关细胞因子的表达而在近视的形成中发挥作用.
揹景 530 nm單色光照射可誘導動物產生近視.Müller細胞作為視網膜上主要的膠質細胞,參與近視的形成,但Müller細胞在單色光誘導的近視形成過程中的具體作用尚不清楚. 目的 研究530 nm單色光照射對體外培養的大鼠視網膜Müller細胞的生物學特徵及近視相關細胞因子錶達的影響,探討Müller細胞在單色光誘導的近視形成中的作用.方法 在自製光照度分彆為125、250、500 lx波長530 nm單色光設備的細胞培養箱中用常規細胞培養基培養永生化大鼠Müller細胞,另用無光照的常規培養法培養細胞作為對照組.細胞培養6、12、24 h後,用四甲基偶氮噻唑藍(MTT)法檢測細胞在波長570 nm處的吸光度(A570)值；培養48 h後用流式細胞儀檢測各組大鼠Müller細胞的細胞週期,比較各組G2期與G1期細胞數的比值；採用逆轉錄PCR(RT-PCR)法檢測各組大鼠Müller細胞中轉化生長因子-β1(TGF-β1)、酪氨痠羥化酶(TH)、誘導型一氧化氮閤成酶(iNOS)和堿性成纖維細胞生長因子(bFGF) mRNA錶達量的改變.採用兩因素方差分析比較各組間大鼠Müller細胞隨不同培養時間的變化增生值的差異,採用單因素方差分析比較各組間不同細胞週期細胞數的變化以及大鼠Müller細胞中各近視相關因子mRNA錶達量的差異. 結果 530 nm單色光照射後,Müller細胞均呈典型的多角形,細胞大小均勻,邊界清晰.與對照組比較,125 lx組、250 lx組大鼠Müller細胞在培養0、6、12、24 h時A570值的差異均無統計學意義(P＞0.05),但500 lx組光照12h、24 h後細胞A570值明顯低于對照組,差異均有統計學意義(P=0.013、0.001).與對照組比較,125 lx組、250 lx組大鼠Müller細胞G2/G1細胞數比值的差異均無統計學意義(P=0.073、0.330),500 lx組G2/G1細胞數比值明顯降低,與對照組、250 lx組比較差異均有統計學意義(P=0.028、0.038).RT-PCR檢測結果顯示,250 lx組TGF-β1 mRNA的錶達明顯高于500 lx組(P=0.006)；125 lx組、250 lx組iNOS mRNA錶達依次上調,250 lx組與對照組比較差異有統計學意義(P=0.001),而500 lx組錶達下調,與對照組比較差異有統計學意義(P=0.000).與對照組比較,125 lx組、250 lx組bFGF mRNA的錶達量均上調,但500 lx組細胞中bFGF mRNA錶達量下調,差異均有統計學意義(P=0.002、0.000、0.005).與對照組比較,250 lx組細胞中TH mRNA的錶達量明顯增加,500 lx組細胞中TH mRNA錶達量明顯下調,差異均有統計學意義(P=0.000、0.001). 結論 光照度＞250 lx的530 nm單色光照射可以通過抑製大鼠Müller細胞的生長,併下調細胞中近視相關細胞因子的錶達而在近視的形成中髮揮作用.
배경 530 nm단색광조사가유도동물산생근시.Müller세포작위시망막상주요적효질세포,삼여근시적형성,단Müller세포재단색광유도적근시형성과정중적구체작용상불청초. 목적 연구530 nm단색광조사대체외배양적대서시망막Müller세포적생물학특정급근시상관세포인자표체적영향,탐토Müller세포재단색광유도적근시형성중적작용.방법 재자제광조도분별위125、250、500 lx파장530 nm단색광설비적세포배양상중용상규세포배양기배양영생화대서Müller세포,령용무광조적상규배양법배양세포작위대조조.세포배양6、12、24 h후,용사갑기우담새서람(MTT)법검측세포재파장570 nm처적흡광도(A570)치；배양48 h후용류식세포의검측각조대서Müller세포적세포주기,비교각조G2기여G1기세포수적비치；채용역전록PCR(RT-PCR)법검측각조대서Müller세포중전화생장인자-β1(TGF-β1)、락안산간화매(TH)、유도형일양화담합성매(iNOS)화감성성섬유세포생장인자(bFGF) mRNA표체량적개변.채용량인소방차분석비교각조간대서Müller세포수불동배양시간적변화증생치적차이,채용단인소방차분석비교각조간불동세포주기세포수적변화이급대서Müller세포중각근시상관인자mRNA표체량적차이. 결과 530 nm단색광조사후,Müller세포균정전형적다각형,세포대소균균,변계청석.여대조조비교,125 lx조、250 lx조대서Müller세포재배양0、6、12、24 h시A570치적차이균무통계학의의(P＞0.05),단500 lx조광조12h、24 h후세포A570치명현저우대조조,차이균유통계학의의(P=0.013、0.001).여대조조비교,125 lx조、250 lx조대서Müller세포G2/G1세포수비치적차이균무통계학의의(P=0.073、0.330),500 lx조G2/G1세포수비치명현강저,여대조조、250 lx조비교차이균유통계학의의(P=0.028、0.038).RT-PCR검측결과현시,250 lx조TGF-β1 mRNA적표체명현고우500 lx조(P=0.006)；125 lx조、250 lx조iNOS mRNA표체의차상조,250 lx조여대조조비교차이유통계학의의(P=0.001),이500 lx조표체하조,여대조조비교차이유통계학의의(P=0.000).여대조조비교,125 lx조、250 lx조bFGF mRNA적표체량균상조,단500 lx조세포중bFGF mRNA표체량하조,차이균유통계학의의(P=0.002、0.000、0.005).여대조조비교,250 lx조세포중TH mRNA적표체량명현증가,500 lx조세포중TH mRNA표체량명현하조,차이균유통계학의의(P=0.000、0.001). 결론 광조도＞250 lx적530 nm단색광조사가이통과억제대서Müller세포적생장,병하조세포중근시상관세포인자적표체이재근시적형성중발휘작용.
Background Research showed that exposure of 530 nm monochromatic light can induce myopia in animal,and retinal Müller cells participate in the formation of myopia.However,the effect and mechanism of retinal Müller cells during the formation of monochromatic light induced-myopia is below understood.Objective This study was to investigate biologic characteristics of rat retina Müller cells and the expression of cell factors in Müller cells after being illuminated by the 530 nm monochromatic light,and discuss the role of the retina Müller cells in myopia induced by monochromatic light.Methods Immortalized rat retinal Müller cells were cultured with DMEM containing 10％ fetal bovine serum in a self-made cell incubator with monochromatic light by adjusting luminance of 530 nm LED source.The cells were exposed to 125,250 and 500 lx luminance respectively for 6,12 and 24 hours,and the cells without light-irradiation were used as control.The growth of the cells under the different light time and different illuminations was described by MTT as the absorbance at the wavelength 570 nm (A570),and cell cycle analysis of Müller cells was performed by flow cytometry 48 hours after cultured,and the expression of transforming growth factor-beta 1 (TGF-β1),tyrosine hydroxylase(TH),inducible nitric oxide synthase(iNOS) and basic fibroblast growth factor(bFGF)in the cells were detected by reverse transcription PCR(RT-PCR),respectively.Results The Müller cells were uniform in size with polygonal shape and defined edges.No statistically significant difference was found in the A570 value in the cells of the 125 lx and 250 lx illuminated groups compared with the control group in various time points(P＞0.05).However,significant lowing was seen in the A570 value in the cells of the 500 lx illuminating for 12 hours and 24 hours in comparison with the control group (P =0.013,0.001).Compared with the control group,the ratio of the number between G2 and G1 phase was not significantly declined in 125 lx,250 lx illuminating for 48 hours (P =0.073,0.330),and the ratio in the 500 lx illuminating group was significantly lower than those in the 250 lx illuminated group and the control group (P =0.028,0.038).RT-PCR revealed that the expression of TGF-β1 mRNA in the cells was higher in the 250 lx illuminated group than that of the 500 lx illuminated group (P=0.006).The expression of iNOS mRNA was gradually upregulated in the 250 lx illuminated group compared with the control group (P =0.001),but that in the 500 lx illuminated group was downregulated (P =0.000).The expression of bFGF mRNA was raised in the 125 lx and 250 lx groups but reduced in the 500 lx group when compared with the control group(P=0.002,0.000,0.005).Also,the expression of TH mRNA was significantly increased in the 250 lx group(P=0.000),but decreased in the 500 lx group(P=0.000,P=0.001).Conclusions The monochromatic light of 530 nm can inhibit the growth of rat Müller cells and downregulate the expression of myopia-related cell factors and therefore exert effect in the formation of myopia.
