Gremlin对人晶状体上皮细胞转分化和细胞外基质合成的影响
Gremlin대인정상체상피세포전분화화세포외기질합성적영향
Effects of Gremlin on transdifferentiation and extracellular matrix synthesis in human lens epithelium cells
  • 万方数据
    中华眼科杂志 중화안과잡지
    Chinese Journal of Ophthalmology
    2015年 8期 597-603 ,共7页

    裴澄%马波%康前雁%秦莉%崔丽珺

    배징%마파%강전안%진리%최려군

    胞间信号肽类和蛋白质类%晶体%上皮细胞%细胞外基质%肌动蛋白类%纤连蛋白类%胶原Ⅰ型 胞間信號肽類和蛋白質類%晶體%上皮細胞%細胞外基質%肌動蛋白類%纖連蛋白類%膠原Ⅰ型
    포간신호태류화단백질류%정체%상피세포%세포외기질%기동단백류%섬련단백류%효원Ⅰ형
    Intercellular signaling peptides and proteins%Lens,crystalline%Epithelial cells%Extracellular matrix%Actins%Fibronectins%Collagen type Ⅰ
    目的 探讨细胞因子Gremlin对体外培养的人晶状体上皮细胞(HLEC)转分化和细胞外基质合成的影响.方法 实验研究.采用不同浓度Gremlin(0、100、200、400 μg/L)对体外培养的HLEC诱导24 h,倒置显微镜下观察细胞形态的变化;用200 μg/L Gremlin诱导HLEC不同时间(0、12、24、48、72 h),实时PCR和Western-blot方法检测转分化标志性蛋白α平滑肌肌动蛋白(α-SMA)、细胞外基质主要成分纤维连接蛋白(Fn)和Ⅰ型胶原纤维(COL-1)的表达.特异性地抑制Gremlin的表达(Gremlin.siRNA)对1.0 μg/L转化生长因子-β2 (TGF-β2)诱导HLEC不同时间(0、12、24、48、72 h)α-SMA 、Fn和COL-1蛋白和基因表达的影响.α-SMA 、Fn和COL-1蛋白和基因的表达与对照组的总体比较采用单因素及双因素方差分析,组间两两比较采用Turkey HSD检验及LSD-t检验.结果 正常HLEC为多角形贴壁细胞,用不同浓度Gremlin诱导24 h后,细胞变为长梭形,细胞间隙变大,呈纤维细胞样改变.细胞纤维样改变程度与Gremlin浓度相关.Gremlin可以诱导HLEC表达α-SMA、Fn和COL-1,并且随着时间的延长(0、12、24、48、72 h),α-SMA、Fn、COL-1 mRNA (α-SMA mRNA的表达:1.00±0.00,1.62±0.57,3.40±0.83,5.90±0.49,7.97±0.91;F=61.64,P＜0.05,q=6.43,13.13,18.66,P＜0.05;Fn mRNA的表达:1.00±0.00,3.26±0.23,5.86±0.90,10.17±2.16,12.89± 1.63;F=42.03,P＜0.05,q=6.45,12.18,15.79,P＜0.05;COL-1 mRNA的表达:1.00±0.00,1.78±0.88,6.80±0.44,12.7±2.46,21.2±3.66;F=51.79,P＜0.05,q=4.97,10.08,17.26,P＜0.05)和蛋白(α-SMA蛋白的表达:0.13±0.02,0.26±0.02,0.29±0.09,0.47±0.06,0.68±0.05;F=45.14,q=5.11,10.67,17.40,P＜0.05;Fn蛋白的表达:0.16±0.04,0.26±0.07,0.65±0.03,0.82±0.04,0.73±0.02;F=144.4,q=20.09,26.78,23.12,P＜0.05;COL-1蛋白的表达:0.11±0.02,0.23±0.09,0.41±0.05,0.61±0.03,0.74±0.03;F=75.47,q=9.99,16.60,21.07,P＜0.05)表达均增强,差异有统计学意义.特异性的阻断Gremlin的表达后,1.0 μg/L TGF-[2诱导人晶状体上皮细胞α-SMA、Fn、COL-1 mRNA (α-SMA mRNA的表达:F组间=81.89,P＜0.05,t=3.234,4.346,10.35;t=2.252,7.272,19.11,P＜O.05;Fn mRNA的表达:F组间=83.61,P＜0.05,t=2.538,8.202,11.99;t=6.316,7.304,14.80,P＜0.05;COL-1 mRNA的表达:F组间=73.64,P＜0.05,t=3.385,7.942,11.64;t=4.794,9.006,13.75,P＜0.05;GremlinmRNA的表达:F组间=46.11,P＜0.05,t=5.08,7.24,8.27;t=6.27,8.27,12.14,P＜0.05)和蛋白(α-SMA蛋白的表达:F组间=129.6,P＜0.05,t=4.34,4.85;3.83,4.34;13.03,14.82,P＜0.05;Fn蛋白的表达:F组间=26.18,P＜0.05,t=5.68,5.95;3.10,4.06;4.19,4.73,P＜0.05;COL-1蛋白的表达:F组间=41.37,P＜0.05,t=6.93,5.51;7.82,6.93;8.71,7.64,P＜0.05;Gremlin蛋白的表达:F组间=59.52,P＜0.05,t=2.24,3.49;5.74,6.23;6.98,9.98,P＜0.05)的表达较对照组明显减少.结论 Gremlin可以时间依赖性地诱导HLEC表达α-SMA、Fn和COL-1,特异性地抑制Gremlin的表达可以在较长时间内有效地抑制TGF-β2对HLEC转分化和细胞外基质合成的作用.
    목적 탐토세포인자Gremlin대체외배양적인정상체상피세포(HLEC)전분화화세포외기질합성적영향.방법 실험연구.채용불동농도Gremlin(0、100、200、400 μg/L)대체외배양적HLEC유도24 h,도치현미경하관찰세포형태적변화;용200 μg/L Gremlin유도HLEC불동시간(0、12、24、48、72 h),실시PCR화Western-blot방법검측전분화표지성단백α평활기기동단백(α-SMA)、세포외기질주요성분섬유련접단백(Fn)화Ⅰ형효원섬유(COL-1)적표체.특이성지억제Gremlin적표체(Gremlin.siRNA)대1.0 μg/L전화생장인자-β2 (TGF-β2)유도HLEC불동시간(0、12、24、48、72 h)α-SMA 、Fn화COL-1단백화기인표체적영향.α-SMA 、Fn화COL-1단백화기인적표체여대조조적총체비교채용단인소급쌍인소방차분석,조간량량비교채용Turkey HSD검험급LSD-t검험.결과 정상HLEC위다각형첩벽세포,용불동농도Gremlin유도24 h후,세포변위장사형,세포간극변대,정섬유세포양개변.세포섬유양개변정도여Gremlin농도상관.Gremlin가이유도HLEC표체α-SMA、Fn화COL-1,병차수착시간적연장(0、12、24、48、72 h),α-SMA、Fn、COL-1 mRNA (α-SMA mRNA적표체:1.00±0.00,1.62±0.57,3.40±0.83,5.90±0.49,7.97±0.91;F=61.64,P＜0.05,q=6.43,13.13,18.66,P＜0.05;Fn mRNA적표체:1.00±0.00,3.26±0.23,5.86±0.90,10.17±2.16,12.89± 1.63;F=42.03,P＜0.05,q=6.45,12.18,15.79,P＜0.05;COL-1 mRNA적표체:1.00±0.00,1.78±0.88,6.80±0.44,12.7±2.46,21.2±3.66;F=51.79,P＜0.05,q=4.97,10.08,17.26,P＜0.05)화단백(α-SMA단백적표체:0.13±0.02,0.26±0.02,0.29±0.09,0.47±0.06,0.68±0.05;F=45.14,q=5.11,10.67,17.40,P＜0.05;Fn단백적표체:0.16±0.04,0.26±0.07,0.65±0.03,0.82±0.04,0.73±0.02;F=144.4,q=20.09,26.78,23.12,P＜0.05;COL-1단백적표체:0.11±0.02,0.23±0.09,0.41±0.05,0.61±0.03,0.74±0.03;F=75.47,q=9.99,16.60,21.07,P＜0.05)표체균증강,차이유통계학의의.특이성적조단Gremlin적표체후,1.0 μg/L TGF-[2유도인정상체상피세포α-SMA、Fn、COL-1 mRNA (α-SMA mRNA적표체:F조간=81.89,P＜0.05,t=3.234,4.346,10.35;t=2.252,7.272,19.11,P＜O.05;Fn mRNA적표체:F조간=83.61,P＜0.05,t=2.538,8.202,11.99;t=6.316,7.304,14.80,P＜0.05;COL-1 mRNA적표체:F조간=73.64,P＜0.05,t=3.385,7.942,11.64;t=4.794,9.006,13.75,P＜0.05;GremlinmRNA적표체:F조간=46.11,P＜0.05,t=5.08,7.24,8.27;t=6.27,8.27,12.14,P＜0.05)화단백(α-SMA단백적표체:F조간=129.6,P＜0.05,t=4.34,4.85;3.83,4.34;13.03,14.82,P＜0.05;Fn단백적표체:F조간=26.18,P＜0.05,t=5.68,5.95;3.10,4.06;4.19,4.73,P＜0.05;COL-1단백적표체:F조간=41.37,P＜0.05,t=6.93,5.51;7.82,6.93;8.71,7.64,P＜0.05;Gremlin단백적표체:F조간=59.52,P＜0.05,t=2.24,3.49;5.74,6.23;6.98,9.98,P＜0.05)적표체교대조조명현감소.결론 Gremlin가이시간의뢰성지유도HLEC표체α-SMA、Fn화COL-1,특이성지억제Gremlin적표체가이재교장시간내유효지억제TGF-β2대HLEC전분화화세포외기질합성적작용.
    Objective To explore the effects of Gremlin on transdifferentiation and extracellular matrix synthesis in cultured human lens epithelium cells (HLEC).Methods This is an experimental research.HLEC were incubated with different concentrations of Gremlin (0,100,200 and 400 μg/L) for 24 h.The morphological changes of HLEC were observed by inverted microscope.Real-time PCR and Western-Blot were used to evaluate the expression of α-smooth muscle actin (α-SMA) (as a landmark protein of epithelial mesenchymal transition),fibronectin (Fn) and collagen type 1 (COL-1) (as major components of extracellular matrix) after stimulation with different time (0,12,24,48,72 h) by 200 μg/L Gremlin.The same parameters were observed in Gremlin.siRNA transfected HLEC which treated with 1.0 μg/L TGF-β2.α-SMA,Fn and COL-1 protein and mRNA expressions comparison with control group were analyzed using one-way and twoway ANOVA,while the difference between groups were compared using Turkey HSD and LSD-t test.Results The normal morphology of HLEC showed polygonal and anchorage-dependent.After the incubation of different concentrations of Gremlin for 24 h,morphological feature of HLEC were changed from monolayer and polygonal to multilayer and spindle fibroblast-like cells,and the intercellular space widened.The expression of α-SMA,Fn and COL-1 were increased with prolonging of Gremlin treatment time (α-SMA gene induction:1.00±0.00,1.62±0.57,3.40±0.83,5.90±0.49,7.97±0.91;F=61.64,P＜0.05,q=6.43,13.13,18.66,P＜0.05;Fn gene induction:1.00±0.00,3.26±0.23,5.86±0.90,10.17±2.16,12.89± 1.63;F=42.03,P＜0.05,q=6.45,12.18,15.79,P＜0.05;COL-1 gene induction:1.00±0.00,1.78±0.88,6.80±0.44,12.76±2.46,21.12±3.66;F=51.79,P＜0.05,q=4.97,10.08,17.26,P＜0.05) (α-SMA protein expression:0.13±0.02,0.26±0.02,0.29±0.09,0.47±0.06,0.68±0.05;F=45.14,q=5.1 1,10.67,17.40,P＜0.05;Fn protein expression:0.16±0.04,0.26±0.07,0.65±0.03,0.82±0.04,0.73±0.02;F=144.4,q=20.09,26.78,23.12,P＜0.05;COL-1 protein expression:0.1 1 ±0.02,0.23±0.09,0.41 ±0.05,0.61 ±0.03,0.74±0.03;F=75.47,q=9.99,16.60,21.07,P＜0.05).Gremlin.siRNA transfection effectively suppressed TGF-β2-induced expression of α-SMA,Fn,and COL-Ⅰ (α-SMA gene induction:F=81.89,P＜0.05,t=3.234,4.346,10.35;t=2.252,7.272,19.11,P＜0.05;Fn gene induction:F=83.61,P＜0.05,t=2.538,8.202,11.99;t=6.316,7.304,14.80,P＜0.05;COL-1 gene induction:F=73.64,P＜0.05,t=3.385,7.942,11.64;t=4.794,9.006,13.75,P＜0.05;Gremlin gene induction:F=46.11,P＜0.05,t=5.08,7.24,8.27;t=6.27,8.27,12.14,P＜0.05) (α-SMA protein expression:F=129.6,P＜0.05,t=4.34,4.85;3.83,4.34;13.03,14.82,P＜0.05;Fn protein expression:F=26.18,P＜0.05,t=5.68,5.95;3.10,4.06;4.19,4.73,P＜0.05;COL-1 protein expression:F=41.37,P＜0.05,t=6.93,5.51;7.82,6.93;8.71,7.64,P＜0.05;Gremlin protein expression:F=59.52,P＜0.05,t=2.24,3.49;5.74,6.23;6.98,9.98,P＜0.05).Conclusions Gremlin could induce HLEC to express α-SMA,Fn and COL-1 in a time-dependent manner and promote transdifferentiation and extracellular matrix synthesis.Specifically silencing the expression of Gremlin could effectively block the TGF-β2-induced EMT and ECM synthesis in HLEC.
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