中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2012年
1期
41-45
,共5页
向建南%张桂兰%张海江%王国华%霍鸣
嚮建南%張桂蘭%張海江%王國華%霍鳴
향건남%장계란%장해강%왕국화%곽명
晶状体上皮细胞%转化生长因子%核心蛋白多糖%后囊膜混浊
晶狀體上皮細胞%轉化生長因子%覈心蛋白多糖%後囊膜混濁
정상체상피세포%전화생장인자%핵심단백다당%후낭막혼탁
Lens epithelial cell%Transforming growth factor%Decorin%Posterior capsular opacification
背景 研究发现,白内障囊外摘出术后组织修复反应可诱导房水中活性转化生长因子β(TGF-β)的增加,残留的晶状体上皮细胞(LECs)移行、分化,细胞外基质沉积,引起上皮-间质转化,导致后囊膜混浊(PCO)的发生.寻求有效的抑制LECs增生的药物对于临床上防治PCO的发生具有重要意义. 目的 探讨核心蛋白多糖( decorin)对兔LECs增生的抑制作用及其剂量-效应与时间-效应的关系. 方法 兔LECs细胞株进行培养和传代,将处于指数生长期的细胞以8×106个/L密度接种于96孔板,将0.1、1.0、10.0 mg/Ldecorin分别加入培养基中培养24、48、72 h,加入体积分数0.1％ DMSO培养的细胞为阳性对照组,常规培养基培养的细胞为空白对照组.MTT比色法分别测定不同质量浓度的decorin作用于LECs不同时间后的细胞生长抑制率；采用流式细胞技术测定各组细胞的细胞周期,用ELISA法检测各组培养液上清中TGF-β的水平,逆转录聚合酶链反应(RT-PCR)法测定TGF-βmRNA在LECs中的表达,免疫细胞化学法观察α-平滑肌肌动蛋白(α-SMA)的表达.结果 ELISA检测结果表明,各组培养基上清液中TGF-β表达量的差异有统计学意义(F=39.24,P=0.03),不同质量浓度组TGF-β水平均明显低于空白对照组,差异有统计学意义(P＜0.01),1.0 mg/L、10.0 mg/L decorin组TGF-β水平均明显低于0.1 mg/L decorin组(P＜0.05).MTT比色法结果显示,≥1.0 mg/L decorin组LECs增生的抑制率明显高于空白对照组,各质量浓度decorin组药物作用48 h和72 h后LECs增生的抑制率明显高于24 h的值,药物作用72 h后LECs增生的抑制率明显高于48 h的值,差异均有统计学意义(P＜0.05),各质量浓度decorin组G0/G1期LECs所占比例均较空白对照组明显增加,差异均有统计学意义(P＜0.05).RT-PCR检测显示,TGF-β mRNA的表达随着decorin质量浓度的升高而降低.免疫组织化学染色表明,10.0 mg/L decorin组α-SMA在LECs中的表达明显弱于空白对照组. 结论 Decorin可以抑制LECs的增生,也可以诱导LECs凋亡,其效应呈明显的剂量和时间依赖性,有望成为最具潜力的防治PCO的药物之一.
揹景 研究髮現,白內障囊外摘齣術後組織脩複反應可誘導房水中活性轉化生長因子β(TGF-β)的增加,殘留的晶狀體上皮細胞(LECs)移行、分化,細胞外基質沉積,引起上皮-間質轉化,導緻後囊膜混濁(PCO)的髮生.尋求有效的抑製LECs增生的藥物對于臨床上防治PCO的髮生具有重要意義. 目的 探討覈心蛋白多糖( decorin)對兔LECs增生的抑製作用及其劑量-效應與時間-效應的關繫. 方法 兔LECs細胞株進行培養和傳代,將處于指數生長期的細胞以8×106箇/L密度接種于96孔闆,將0.1、1.0、10.0 mg/Ldecorin分彆加入培養基中培養24、48、72 h,加入體積分數0.1％ DMSO培養的細胞為暘性對照組,常規培養基培養的細胞為空白對照組.MTT比色法分彆測定不同質量濃度的decorin作用于LECs不同時間後的細胞生長抑製率；採用流式細胞技術測定各組細胞的細胞週期,用ELISA法檢測各組培養液上清中TGF-β的水平,逆轉錄聚閤酶鏈反應(RT-PCR)法測定TGF-βmRNA在LECs中的錶達,免疫細胞化學法觀察α-平滑肌肌動蛋白(α-SMA)的錶達.結果 ELISA檢測結果錶明,各組培養基上清液中TGF-β錶達量的差異有統計學意義(F=39.24,P=0.03),不同質量濃度組TGF-β水平均明顯低于空白對照組,差異有統計學意義(P＜0.01),1.0 mg/L、10.0 mg/L decorin組TGF-β水平均明顯低于0.1 mg/L decorin組(P＜0.05).MTT比色法結果顯示,≥1.0 mg/L decorin組LECs增生的抑製率明顯高于空白對照組,各質量濃度decorin組藥物作用48 h和72 h後LECs增生的抑製率明顯高于24 h的值,藥物作用72 h後LECs增生的抑製率明顯高于48 h的值,差異均有統計學意義(P＜0.05),各質量濃度decorin組G0/G1期LECs所佔比例均較空白對照組明顯增加,差異均有統計學意義(P＜0.05).RT-PCR檢測顯示,TGF-β mRNA的錶達隨著decorin質量濃度的升高而降低.免疫組織化學染色錶明,10.0 mg/L decorin組α-SMA在LECs中的錶達明顯弱于空白對照組. 結論 Decorin可以抑製LECs的增生,也可以誘導LECs凋亡,其效應呈明顯的劑量和時間依賴性,有望成為最具潛力的防治PCO的藥物之一.
배경 연구발현,백내장낭외적출술후조직수복반응가유도방수중활성전화생장인자β(TGF-β)적증가,잔류적정상체상피세포(LECs)이행、분화,세포외기질침적,인기상피-간질전화,도치후낭막혼탁(PCO)적발생.심구유효적억제LECs증생적약물대우림상상방치PCO적발생구유중요의의. 목적 탐토핵심단백다당( decorin)대토LECs증생적억제작용급기제량-효응여시간-효응적관계. 방법 토LECs세포주진행배양화전대,장처우지수생장기적세포이8×106개/L밀도접충우96공판,장0.1、1.0、10.0 mg/Ldecorin분별가입배양기중배양24、48、72 h,가입체적분수0.1％ DMSO배양적세포위양성대조조,상규배양기배양적세포위공백대조조.MTT비색법분별측정불동질량농도적decorin작용우LECs불동시간후적세포생장억제솔；채용류식세포기술측정각조세포적세포주기,용ELISA법검측각조배양액상청중TGF-β적수평,역전록취합매련반응(RT-PCR)법측정TGF-βmRNA재LECs중적표체,면역세포화학법관찰α-평활기기동단백(α-SMA)적표체.결과 ELISA검측결과표명,각조배양기상청액중TGF-β표체량적차이유통계학의의(F=39.24,P=0.03),불동질량농도조TGF-β수평균명현저우공백대조조,차이유통계학의의(P＜0.01),1.0 mg/L、10.0 mg/L decorin조TGF-β수평균명현저우0.1 mg/L decorin조(P＜0.05).MTT비색법결과현시,≥1.0 mg/L decorin조LECs증생적억제솔명현고우공백대조조,각질량농도decorin조약물작용48 h화72 h후LECs증생적억제솔명현고우24 h적치,약물작용72 h후LECs증생적억제솔명현고우48 h적치,차이균유통계학의의(P＜0.05),각질량농도decorin조G0/G1기LECs소점비례균교공백대조조명현증가,차이균유통계학의의(P＜0.05).RT-PCR검측현시,TGF-β mRNA적표체수착decorin질량농도적승고이강저.면역조직화학염색표명,10.0 mg/L decorin조α-SMA재LECs중적표체명현약우공백대조조. 결론 Decorin가이억제LECs적증생,야가이유도LECs조망,기효응정명현적제량화시간의뢰성,유망성위최구잠력적방치PCO적약물지일.
Background Researches found that the posterior capsular opacification (PCO) after lensextraction is associated with the elevation of the transforming growth factor-β(TGF-β).To seek the drug for inhibitingproliferation of lens epithelial cells (LECs) is crucial in the treatment and prevention of PCO. Objective Thisstudy was to investigate the preventing effects of decorin on the proliferation of LECs. Methods Rabbit LECs wascultured and passaged.The LECs in growth phase were incubated in 96 well plate at the density of 8×106/L.Decorinwith the concentrations 0.1,1.0,10.0 mg/L was added into the medium for 24,48 and 72 hours respectively.0.1％DMSO was used at the same way as positive control group,and the regular cultured cells worked as blank controlgroup.The inhibitory rates of different concentrations of decorin on the growth of LECs were detected by MTT at 24,48and 72 hours after addition of decorin.The percentage of LECs in different cell cycles in various groups was assayedusing flow cytometry.TGF-β level in medium suspension was detected using ELISA.The expression of TGF-β mRNA in LECs was checked by RT-PCR,and α-SMA expression in LECs was determined using immunochemistry. Results ELISA assay showed a statistical difference in the TGF-β levels of different groups (F=39.24,P=0.03 ).The TGF-β levels in 1.0,10.0 mg/L decorin groups were significantly decreased in comparison with blank control group (P＜0.01) and 0.1 mg/L decorin group (P＜0.05 ).The inhibitory rates of decorin in the concentrations of ≥ 1.0 mg/L on the growth of LECs were higher than the blank control group,and those in various concentrations of decorin groups were considerably lower in 24 hours compared with 48 and 72 hours ( P＜0.05 ) and so was the 48 hours compared with 72 hours (P＜0.05 ).The percentages of LECs in G0/G1 phase were ascent in 0.1,1.0 and 10.0 mg/L decorin groups in comparison with G2/M and S phase (P＜0.05).Immunochemistry revealed the weak expression of α-SMA in various decorin groups in comparison with control group. Conclusions Decorin can effectively inhibit LECs growth and induce LECs apoptosis in concentration- and time-dependent manner.It is suggested that decorin can be used in the prevention and treatment of after cataract.
