中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2013年
4期
347-351
,共5页
雷帕霉素%转化生长因子-β2%人晶状体上皮细胞%上皮细胞-肌成纤维细胞转化
雷帕黴素%轉化生長因子-β2%人晶狀體上皮細胞%上皮細胞-肌成纖維細胞轉化
뢰파매소%전화생장인자-β2%인정상체상피세포%상피세포-기성섬유세포전화
Rapamycin%Transforming growth factor-β2%Human lens epithelial cell%Epithelial-myofibroblast transition
背景 转化生长因子-β2(TGF-β2)诱导的人晶状体上皮细胞(LECs)上皮细胞-肌成纤维细胞转化(EMT)过程是后发性白内障(PCO)的主要发病机制,因此寻求有效抑制这一作用的药物对于防治PCO有重要意义. 目的 观察雷帕霉素(RAPA)对人LECs增生和TGF-β2诱导的EMT的抑制作用. 方法 采用含质量分数10％胎牛血清的DMEM高糖细胞培养液对人LECs系SRA01/04进行体外培养,换以无血清培养液培养后分别用TGF-β2(5 mg/L)、TGF-β2+ 10 mg/L RAPA、TGF-β2+ 100 mg/L RAPA、TGF-β2+ 1000 mg/LRAPA、TGF-β2+10 000 mg/L RAPA共培养SRA01/04 72 h,仅用无血清培养液培养的SRA01/04作为对照组.采用MTT法检测各组SRA01/04的吸光度(A490)值以评估SRA01/04的增生情况,并计算RAPA对细胞生长的抑制率.各组细胞作用48 h后,采用逆转录PCR(RT-PCR)法和Western blot法分别检测LECs ETM的标志物-α平滑肌肌动蛋白(α-SMA)和上皮性钙黏附蛋白(E-cad)的表达.选取TGF-β2 +400 mg/L RAPA分别作用于培养的SRA01/04 24、48、72 h,采用Western blot法检测SRA01/04中α-SMA、E-cad的表达水平. 结果 MTT法检测结果显示,对照组、TGF-β2组、TGF-β2+10 mg/L RAPA组、TGF-β2+ 100 mg/L RAPA组、TGF-β2+1000 mg/L RAPA组和TGF-β2+10 000 mg/L RAPA组SRA01/04的A490值分别为0.680±0.020、0.550±0.013、0.480±0.014、0.400±0.011和0.200±0.019,表明随着RAPA质量浓度的增加,SRA01/04增生值降低,差异有统计学意义(F=101.920,P=0.000),且RAPA的抑制率逐渐增加.RT-PCR和Western blot检测结果表明,阴性对照组SRA01/04中α-SMA mRNA(α-SMA mRNA/β-actin mRNA)及其蛋白(α-SMA /β-actin)仅有少量表达,TGF-β2组可见SRA01/04中α-SMA mRNA及其蛋白表达量明显增加,但不同质量浓度的RAPA作用于SR A01/04后,随着RAPA质量浓度的增加,SRA01/04中α-SMA mRNA及蛋白的表达量均逐渐减少,但E-cadmRNA及其蛋白表达逐渐增多,各组间的总体差异均有统计学意义(α-SMA mRNA:F=294.660,P=0.000;α-SMA蛋白:F=346.950,P=0.000;E-cad mRNA:F=264.250,P=0.000;E-cad蛋白:F=317.327,P=0.000).400 mg/L RAPA作用于SRA01/04后,随着作用时间的延长,α-SMA蛋白的表达量逐渐下降,而E-cad蛋白的表达量逐渐增加,差异均有统计学意义(α-SMA:F=693.864,P=0.000;E-cad:F=369.286,P=0.000). 结论 RAPA可抑制体外培养的SRA01/04增生,其作用呈剂量依赖性,此外RAPA具有抑制TGF-β2诱导的LECs ETM作用,其作用呈剂量和时间依赖性.
揹景 轉化生長因子-β2(TGF-β2)誘導的人晶狀體上皮細胞(LECs)上皮細胞-肌成纖維細胞轉化(EMT)過程是後髮性白內障(PCO)的主要髮病機製,因此尋求有效抑製這一作用的藥物對于防治PCO有重要意義. 目的 觀察雷帕黴素(RAPA)對人LECs增生和TGF-β2誘導的EMT的抑製作用. 方法 採用含質量分數10％胎牛血清的DMEM高糖細胞培養液對人LECs繫SRA01/04進行體外培養,換以無血清培養液培養後分彆用TGF-β2(5 mg/L)、TGF-β2+ 10 mg/L RAPA、TGF-β2+ 100 mg/L RAPA、TGF-β2+ 1000 mg/LRAPA、TGF-β2+10 000 mg/L RAPA共培養SRA01/04 72 h,僅用無血清培養液培養的SRA01/04作為對照組.採用MTT法檢測各組SRA01/04的吸光度(A490)值以評估SRA01/04的增生情況,併計算RAPA對細胞生長的抑製率.各組細胞作用48 h後,採用逆轉錄PCR(RT-PCR)法和Western blot法分彆檢測LECs ETM的標誌物-α平滑肌肌動蛋白(α-SMA)和上皮性鈣黏附蛋白(E-cad)的錶達.選取TGF-β2 +400 mg/L RAPA分彆作用于培養的SRA01/04 24、48、72 h,採用Western blot法檢測SRA01/04中α-SMA、E-cad的錶達水平. 結果 MTT法檢測結果顯示,對照組、TGF-β2組、TGF-β2+10 mg/L RAPA組、TGF-β2+ 100 mg/L RAPA組、TGF-β2+1000 mg/L RAPA組和TGF-β2+10 000 mg/L RAPA組SRA01/04的A490值分彆為0.680±0.020、0.550±0.013、0.480±0.014、0.400±0.011和0.200±0.019,錶明隨著RAPA質量濃度的增加,SRA01/04增生值降低,差異有統計學意義(F=101.920,P=0.000),且RAPA的抑製率逐漸增加.RT-PCR和Western blot檢測結果錶明,陰性對照組SRA01/04中α-SMA mRNA(α-SMA mRNA/β-actin mRNA)及其蛋白(α-SMA /β-actin)僅有少量錶達,TGF-β2組可見SRA01/04中α-SMA mRNA及其蛋白錶達量明顯增加,但不同質量濃度的RAPA作用于SR A01/04後,隨著RAPA質量濃度的增加,SRA01/04中α-SMA mRNA及蛋白的錶達量均逐漸減少,但E-cadmRNA及其蛋白錶達逐漸增多,各組間的總體差異均有統計學意義(α-SMA mRNA:F=294.660,P=0.000;α-SMA蛋白:F=346.950,P=0.000;E-cad mRNA:F=264.250,P=0.000;E-cad蛋白:F=317.327,P=0.000).400 mg/L RAPA作用于SRA01/04後,隨著作用時間的延長,α-SMA蛋白的錶達量逐漸下降,而E-cad蛋白的錶達量逐漸增加,差異均有統計學意義(α-SMA:F=693.864,P=0.000;E-cad:F=369.286,P=0.000). 結論 RAPA可抑製體外培養的SRA01/04增生,其作用呈劑量依賴性,此外RAPA具有抑製TGF-β2誘導的LECs ETM作用,其作用呈劑量和時間依賴性.
배경 전화생장인자-β2(TGF-β2)유도적인정상체상피세포(LECs)상피세포-기성섬유세포전화(EMT)과정시후발성백내장(PCO)적주요발병궤제,인차심구유효억제저일작용적약물대우방치PCO유중요의의. 목적 관찰뢰파매소(RAPA)대인LECs증생화TGF-β2유도적EMT적억제작용. 방법 채용함질량분수10％태우혈청적DMEM고당세포배양액대인LECs계SRA01/04진행체외배양,환이무혈청배양액배양후분별용TGF-β2(5 mg/L)、TGF-β2+ 10 mg/L RAPA、TGF-β2+ 100 mg/L RAPA、TGF-β2+ 1000 mg/LRAPA、TGF-β2+10 000 mg/L RAPA공배양SRA01/04 72 h,부용무혈청배양액배양적SRA01/04작위대조조.채용MTT법검측각조SRA01/04적흡광도(A490)치이평고SRA01/04적증생정황,병계산RAPA대세포생장적억제솔.각조세포작용48 h후,채용역전록PCR(RT-PCR)법화Western blot법분별검측LECs ETM적표지물-α평활기기동단백(α-SMA)화상피성개점부단백(E-cad)적표체.선취TGF-β2 +400 mg/L RAPA분별작용우배양적SRA01/04 24、48、72 h,채용Western blot법검측SRA01/04중α-SMA、E-cad적표체수평. 결과 MTT법검측결과현시,대조조、TGF-β2조、TGF-β2+10 mg/L RAPA조、TGF-β2+ 100 mg/L RAPA조、TGF-β2+1000 mg/L RAPA조화TGF-β2+10 000 mg/L RAPA조SRA01/04적A490치분별위0.680±0.020、0.550±0.013、0.480±0.014、0.400±0.011화0.200±0.019,표명수착RAPA질량농도적증가,SRA01/04증생치강저,차이유통계학의의(F=101.920,P=0.000),차RAPA적억제솔축점증가.RT-PCR화Western blot검측결과표명,음성대조조SRA01/04중α-SMA mRNA(α-SMA mRNA/β-actin mRNA)급기단백(α-SMA /β-actin)부유소량표체,TGF-β2조가견SRA01/04중α-SMA mRNA급기단백표체량명현증가,단불동질량농도적RAPA작용우SR A01/04후,수착RAPA질량농도적증가,SRA01/04중α-SMA mRNA급단백적표체량균축점감소,단E-cadmRNA급기단백표체축점증다,각조간적총체차이균유통계학의의(α-SMA mRNA:F=294.660,P=0.000;α-SMA단백:F=346.950,P=0.000;E-cad mRNA:F=264.250,P=0.000;E-cad단백:F=317.327,P=0.000).400 mg/L RAPA작용우SRA01/04후,수착작용시간적연장,α-SMA단백적표체량축점하강,이E-cad단백적표체량축점증가,차이균유통계학의의(α-SMA:F=693.864,P=0.000;E-cad:F=369.286,P=0.000). 결론 RAPA가억제체외배양적SRA01/04증생,기작용정제량의뢰성,차외RAPA구유억제TGF-β2유도적LECs ETM작용,기작용정제량화시간의뢰성.
Background Epithelial-myofibroblast transition (EMT) of human lens epithelial cells (LECs) induced by transforming growth factor-β2 (TGF-β2) is the main mechanism in the pathogenesis of posterior capsular opacification(PCO).Seeking an effective drug capable of inhibiting this process is important for the prevention and treatment of PCO.Objective The purpose of this study was to investigate the inhibitory effect of rapamycin (RAPA)on the proliferation of human LECs and TGF-β2-induced EMT.Methods Human LEC strain(SRA01/04)was cultured in DMEM with high glucose and 10％ fetal bovine serum.The cells were consequently cultured in serumfree DMEM with 5 mg/L TGF-β2,TGF-β2+10 mg/L RAPA,TGF-β2 + 100 mg/L RAPA,TGF-β2 + 1000 mg/L RAPA or TGF-β2 +10 000 mg/L RAPA for 72 hours,and SRA01/04 cultured in serum-free DMEM were used as control.The proliferation rate(A490)of SRA01/04 in the different groups was detected using the MTT assay and the rate of inhibition of RAPA was calculated.The expressions of the α-smooth muscle actin(α-SMA) and E-cadherin(E-cad)mRNA and protein were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot,respectively.The changes in the expression of α-SMA and E-cad in SRA01/04 were evaluated by Western blot 24,48 and 72 hours after TGF-β2 +400 mg/L RAPA treatment.Results The A490 value of SRA01/04 was 0.680±0.020,0.550±0.013,0.480±0.014,0.400±0.011 and 0.200±0.019 in the control group,TGF-β2 group,TGF-β2 + 10 mg/LRAPA group,TGF-β2 + 100 mg/L RAPA group,TGF-β2 + 1000 mg/L RAPA and TGF-β2 + 10 000 mg/L RAPA group,respectively,showing a gradually declining trend in SRA01/04 rate of proliferation with increasing RAPA concentrations (F =101.920,P =0.000).RT-PCR and Western blot assay showed that the relative expression levels of α-SMA mRNA (α-SMA mRNA/β-actin mRNA)and protein (α-SMA/β-actin)in the cells were significantly increased in the TGF-β2 treatment group.However,with exposure to RAPA,the relative expression levels of α-SMA mRNA and protein were significantly lowered with increasing RAPA concentrations,but the expression levels of E-cad mRNA and protein were raised (α-SMA mRNA:F =294.660,P =0.000 ; α-SMA protein:F =346.950,P =0.000 ; E-cad mRNA:F =264.250,P =0.000 ; E-cad protein:F =317.327,P =0.000).In addition,after exposure to 400 mg/L RAPA,the expression levels of α-SMA protein gradually reduced and those of E-cad protein gradually increased with increasing treatment durations,showing significant differences among the different time points (α-SMA:F =693.864,P =0.000 ;E-cad:F=369.286,P =0.000).Conclusions RAPA can inhibit the proliferation of SRA01/04 in vitro and arrest EMT of SRA01/04 induced by TGF-β2 in a dose-and time-dependent manner.
