不同波长的光照射对视网膜色素上皮细胞生长及其分泌生长因子的影响
불동파장적광조사대시망막색소상피세포생장급기분비생장인자적영향
Effects of different wave-length lights on proliferation and secretion of growth factors in human retinal pigment epithelium cells
  • 万方数据
    中华实验眼科杂志 중화실험안과잡지
    CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
    2011年 9期 774-779 ,共6页

    郑晓汾%康玉国%褚仁远

    정효분%강옥국%저인원

    近视%视网膜色素上皮细胞%肝细胞生长因子%碱性成纤维细胞生长因子%转化生长因子-β%光照射 近視%視網膜色素上皮細胞%肝細胞生長因子%堿性成纖維細胞生長因子%轉化生長因子-β%光照射
    근시%시망막색소상피세포%간세포생장인자%감성성섬유세포생장인자%전화생장인자-β%광조사
    Myopia%Retinal pigment epithelial cell%Hepatocyte growth factor%Basic fibroblast growth factor%Transforming growth factor-β%Light exposure
    背景 近视的发病机制是眼科研究的一个热点，近年来发现，由神经视网膜细胞及视网膜色素上皮(RPE)细胞分泌的细胞因子可能参与近视的发病，RPE细胞因子的分泌功能受光照射的影响，而RPE细胞对不同波长光的吸光度(A)值是不一样的。 目的 研究不同波长的光照射对人胚RPE细胞增生及其分泌肝细胞生长因子(HGF)、碱性成纤维细胞生长因子(bFGF)和转化生长因子-β(TGF-β)的影响，从细胞水平和分子生物学水平探讨近视的发病机制。 方法第4～5代人胚RPE细胞置于白光、波长为775 nm的红光及波长为480 nm的蓝光下照射和培养48 h，MTT比色法检测各组人RPE细胞的增生情况，并用透射电子显微镜观察各组传代细胞的超微结构。分别于光照射后12、24、48、72 h收集培养液，采用酶联免疫吸附实验( ELISA)测定培养基中HGF、bFGF和TGF-β的质量浓度。用逆转录聚合酶链反应(RT-PCR)检测不同波长光照射24 h后RPE细胞中HGF mRNA的表达水平，分析蓝光、红光、白光照射对体外培养RPE细胞的影响。结果蓝光组、红光组、白光组和对照组RPE细胞的A490值分别为0.0218+0.0014、0.0353±0.0025、0.0371±0.0024和0.0445+0.0046。4个组中RPE细胞的增生速度明显不同，差异有统计学意义(F=12.579，P＜0.05)，蓝光组和红光组A490值均明显低于对照组，差异均有统计学意义(t=2.043、t=2.024,P＜0.05)。ELISA结果显示人胚RPE细胞可以分泌HGF、bFGF和TGF-β，在受到不同波长的光照射后，不同时间点HGF、bFGF和TGF-β的分泌量不同，随着时间的变化，4个组HGF、TGF-β的质量浓度均逐渐升高，二者在72 h和48 h均高于12 h(P＜0.05)，红光组与蓝光组更为明显，与对照组比较差异均有统计学意义(P＜0.05)。各波长组bFGF的质量浓度随着光照时间的延长在RPE细胞中的表达均逐渐降低，72 h与12h时相比差异均有统计学意义(P＜0.05)。RT-PCR结果显示，不同波长光照射后RPE细胞中HGF mRNA的表达水平不同，蓝光下培养的RPE细胞HGF mRNA表达量最少，白光组、红光组和蓝光组HGF mRNA的表达量(A490)与对照组比较差异均有统计学意义( P＜0.05)。透射电子显微镜下红光组、白光组人胚RPE细胞与对照组相比超微结构均无明显变化，但蓝光组RPE细胞核染色质稀疏变淡，细胞器减少，细胞界膜不清或消失。结论不同波长的光照射可影响人RPE细胞的增生及其分泌HGF、bFGF和TGF-β的功能，提示近视的形成可能与不同波长的光相互作用有关。
    배경 근시적발병궤제시안과연구적일개열점，근년래발현，유신경시망막세포급시망막색소상피(RPE)세포분비적세포인자가능삼여근시적발병，RPE세포인자적분비공능수광조사적영향，이RPE세포대불동파장광적흡광도(A)치시불일양적。 목적 연구불동파장적광조사대인배RPE세포증생급기분비간세포생장인자(HGF)、감성성섬유세포생장인자(bFGF)화전화생장인자-β(TGF-β)적영향，종세포수평화분자생물학수평탐토근시적발병궤제。 방법제4～5대인배RPE세포치우백광、파장위775 nm적홍광급파장위480 nm적람광하조사화배양48 h，MTT비색법검측각조인RPE세포적증생정황，병용투사전자현미경관찰각조전대세포적초미결구。분별우광조사후12、24、48、72 h수집배양액，채용매련면역흡부실험( ELISA)측정배양기중HGF、bFGF화TGF-β적질량농도。용역전록취합매련반응(RT-PCR)검측불동파장광조사24 h후RPE세포중HGF mRNA적표체수평，분석람광、홍광、백광조사대체외배양RPE세포적영향。결과람광조、홍광조、백광조화대조조RPE세포적A490치분별위0.0218+0.0014、0.0353±0.0025、0.0371±0.0024화0.0445+0.0046。4개조중RPE세포적증생속도명현불동，차이유통계학의의(F=12.579，P＜0.05)，람광조화홍광조A490치균명현저우대조조，차이균유통계학의의(t=2.043、t=2.024,P＜0.05)。ELISA결과현시인배RPE세포가이분비HGF、bFGF화TGF-β，재수도불동파장적광조사후，불동시간점HGF、bFGF화TGF-β적분비량불동，수착시간적변화，4개조HGF、TGF-β적질량농도균축점승고，이자재72 h화48 h균고우12 h(P＜0.05)，홍광조여람광조경위명현，여대조조비교차이균유통계학의의(P＜0.05)。각파장조bFGF적질량농도수착광조시간적연장재RPE세포중적표체균축점강저，72 h여12h시상비차이균유통계학의의(P＜0.05)。RT-PCR결과현시，불동파장광조사후RPE세포중HGF mRNA적표체수평불동，람광하배양적RPE세포HGF mRNA표체량최소，백광조、홍광조화람광조HGF mRNA적표체량(A490)여대조조비교차이균유통계학의의( P＜0.05)。투사전자현미경하홍광조、백광조인배RPE세포여대조조상비초미결구균무명현변화，단람광조RPE세포핵염색질희소변담，세포기감소，세포계막불청혹소실。결론불동파장적광조사가영향인RPE세포적증생급기분비HGF、bFGF화TGF-β적공능，제시근시적형성가능여불동파장적광상호작용유관。
    Background The study of myopia development is always the hotspot worldwide. Recently,scientist found that some growth factor secreted by retinal nerve epithelium cells and retinal pigment epithelium(RPE)cells are associated with the development of myopia. Whenever, the absorption of RPE cells to different wave-length lights is different. ObjectiveThis study was to investigate the effects of different wave-lengths lights on the proliferation of human RPE cells, and explore the influence of different wave-lengths lights on RPE cells secreting hepatocyte growth factor(HGF) ,basic fibroblast growth factor(bFGF) and transforming growth factor-β(TGF-β).Methods The fourth to fifth passages of human embryonic RPE cells were exposed to blue light( λ =480 nm),red light( λ =775 nm) and white light. The cells of control group were harvested in normal condition. The proliferation and growth of RPE cells were assayed by MTT,and the ultrastructure of cells was examined under the transmission electron microscopy at 48 hours after light exposure of RPE cells. Enzyme linked immunosorbent assay(ELISA) was adopted to determine the concentrations of HGF,bFGF and TGF-β in the culture medium in 12,24,48,72 hours. The expression of HGF mRNA in RPE cells was detected by RT-PCR. This study was approved by Ethic committee of Fudan University. Results The A490 values of the cells exposed to blue light,red light,white light and white light were 0. 0218±0. 0014 ;0. 0353±0. 0025 ;0. 0371 ±0. 0024 and 0. 0445 +0. 0046 respectively with the significant difference among 4 groups ( F =12. 579, P＜0.05 ), and A490 value in blue light group, red light group were significantly lower than that of the control group ( t =2.043 ; t =2.024, P＜0.05 ). ELISA showed that the concentrations of HGF and TGF-β in culture medium were evidently elevated as the prolongation of light exposure in various light exposure groups in 72 hours(HGF) and 48 hours(TGF-β) compared with 12 hours with a predominating rise in the control group. The statistically significant differences were found in the concentrations of HGF and TGF-β between control group and blue light group or red light group in the( all P＜0. 05 ). The bFGF level was decreased with the time increase of various light exposure with the significant differences in 72 hours compared with 12 hours( P＜0.05 ). RT-PCR revealed the considerable difference about expression of HGF mRNA in RPE cells among these four groups( P＜0. 05 ), and the lest expression in HGF mRNA was in the blue light group compared with control group( t =3. 972,P＜0.05 ). Thinning of the chromatin, decreasing of organelle and loss of cellular membrane were seen in the blue light group, but no obvious change of ultrastructure of human embryo RPE cells was found in the ret and white light groups. Conclusions The irradiation of different wave-length light can effect the growth and proliferation and secretion of HGF,bFGF and TGFβ in human RPE cells in vitro,implying myopia formation is associated to exposure of different wave-length light.
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