中华眼科杂志
中華眼科雜誌
중화안과잡지
Chinese Journal of Ophthalmology
2013年
9期
822-828
,共7页
裴澄%马波%康前雁%秦莉%张小玲%崔丽珺
裴澄%馬波%康前雁%秦莉%張小玲%崔麗珺
배징%마파%강전안%진리%장소령%최려군
转化生长因子β2%晶体%上皮细胞%结缔组织生长因子%肌动蛋白类%胶原Ⅰ型%细胞外基质
轉化生長因子β2%晶體%上皮細胞%結締組織生長因子%肌動蛋白類%膠原Ⅰ型%細胞外基質
전화생장인자β2%정체%상피세포%결체조직생장인자%기동단백류%효원Ⅰ형%세포외기질
Transforming growth factor beta 2%Lens,crystalline%Epithelial cells%Connective tissue growth factor%Actins%Collagen type Ⅰ%Extracellular matrix
目的 探讨转化生长因子β2(TGF-β2)对体外培养的人晶状体上皮细胞(HLEC)转分化、细胞外基质(ECM)合成及结缔组织生长因子(CTGF)表达的影响.方法 实验研究.采用不同浓度TGF-β2(0.0、0.1、1.0、10.0μg/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；免疫荧光法检测CTGF、间质转分化标志性蛋白α-平滑肌肌动蛋白(α-SMA)在细胞中的表达时实PCR和Western-blot方法检测CTGF、α-SMA、细胞外基质主要成分Ⅰ型胶原纤维(COL-Ⅰ)和纤维连接蛋白(Fn)的表达.结果 正常HLEC为多角形贴壁细胞,用不同浓度TGF-β2诱导24 h后,细胞由单层多角形变为长梭形,细胞间隙变大,呈纤维细胞样改变.TGF-β2可诱导HLEC表达CTGF,并且随浓度的增加,CTGF蛋白(0.53±0.03、0.73±0.01、0.65±0.03)和mRNA(1.00±0.00、7.18±0.41、12.88±0.45、32.84±1.61)表达均增强,差异有统计学意义(F=77.55,P＜0.05;F=379.0,P＜0.05).TGF-β2能诱导HLEC转分化,浓度依赖性地促进α-SMA蛋白(0.48 ±0.01、0.78 ±0.04、0.69±0.04)和mRNA(1.00 ±0.00、2.30±0.22、3.10±0.21、3.86 ±0.10)的表达,差异有统计学意义(F =62.73,P＜0.05;F=80.22,P＜0.05).不同浓度(0.0、0.1、1.0、10.0 μg/L)TGF-β2从基因和蛋白水平促进HLEC表达COL-Ⅰ和Fn,COL-Ⅰ mRNA的相对值分别为1.00 ±0.00、5.52±0.96、18.31 ±1.20、82.51 ±1.45,Fn mRNA的相对值分别为1.00 ±0.00、2.36±0.25、3.27 ±0.24、4.25±0.24；COL-Ⅰ蛋白表达相对值分别为0.78 ±0.05、1.15±0.11、2.16±0.14,Fn蛋白表达相对值为0.64±0.01、0.95 ±0.02、1.23 ±0.14,其作用呈浓度依赖性(F=1880.52,105.30,P＜0.05;F=64.44,51.81,P＜0.05).结论 TGF-β2可以诱导HLEC表达CTGF,促进HLEC转分化及细胞外基质的合成.
目的 探討轉化生長因子β2(TGF-β2)對體外培養的人晶狀體上皮細胞(HLEC)轉分化、細胞外基質(ECM)閤成及結締組織生長因子(CTGF)錶達的影響.方法 實驗研究.採用不同濃度TGF-β2(0.0、0.1、1.0、10.0μg/L)對體外培養的HLEC誘導24 h,倒置顯微鏡下觀察細胞形態的變化；免疫熒光法檢測CTGF、間質轉分化標誌性蛋白α-平滑肌肌動蛋白(α-SMA)在細胞中的錶達時實PCR和Western-blot方法檢測CTGF、α-SMA、細胞外基質主要成分Ⅰ型膠原纖維(COL-Ⅰ)和纖維連接蛋白(Fn)的錶達.結果 正常HLEC為多角形貼壁細胞,用不同濃度TGF-β2誘導24 h後,細胞由單層多角形變為長梭形,細胞間隙變大,呈纖維細胞樣改變.TGF-β2可誘導HLEC錶達CTGF,併且隨濃度的增加,CTGF蛋白(0.53±0.03、0.73±0.01、0.65±0.03)和mRNA(1.00±0.00、7.18±0.41、12.88±0.45、32.84±1.61)錶達均增彊,差異有統計學意義(F=77.55,P＜0.05;F=379.0,P＜0.05).TGF-β2能誘導HLEC轉分化,濃度依賴性地促進α-SMA蛋白(0.48 ±0.01、0.78 ±0.04、0.69±0.04)和mRNA(1.00 ±0.00、2.30±0.22、3.10±0.21、3.86 ±0.10)的錶達,差異有統計學意義(F =62.73,P＜0.05;F=80.22,P＜0.05).不同濃度(0.0、0.1、1.0、10.0 μg/L)TGF-β2從基因和蛋白水平促進HLEC錶達COL-Ⅰ和Fn,COL-Ⅰ mRNA的相對值分彆為1.00 ±0.00、5.52±0.96、18.31 ±1.20、82.51 ±1.45,Fn mRNA的相對值分彆為1.00 ±0.00、2.36±0.25、3.27 ±0.24、4.25±0.24；COL-Ⅰ蛋白錶達相對值分彆為0.78 ±0.05、1.15±0.11、2.16±0.14,Fn蛋白錶達相對值為0.64±0.01、0.95 ±0.02、1.23 ±0.14,其作用呈濃度依賴性(F=1880.52,105.30,P＜0.05;F=64.44,51.81,P＜0.05).結論 TGF-β2可以誘導HLEC錶達CTGF,促進HLEC轉分化及細胞外基質的閤成.
목적 탐토전화생장인자β2(TGF-β2)대체외배양적인정상체상피세포(HLEC)전분화、세포외기질(ECM)합성급결체조직생장인자(CTGF)표체적영향.방법 실험연구.채용불동농도TGF-β2(0.0、0.1、1.0、10.0μg/L)대체외배양적HLEC유도24 h,도치현미경하관찰세포형태적변화；면역형광법검측CTGF、간질전분화표지성단백α-평활기기동단백(α-SMA)재세포중적표체시실PCR화Western-blot방법검측CTGF、α-SMA、세포외기질주요성분Ⅰ형효원섬유(COL-Ⅰ)화섬유련접단백(Fn)적표체.결과 정상HLEC위다각형첩벽세포,용불동농도TGF-β2유도24 h후,세포유단층다각형변위장사형,세포간극변대,정섬유세포양개변.TGF-β2가유도HLEC표체CTGF,병차수농도적증가,CTGF단백(0.53±0.03、0.73±0.01、0.65±0.03)화mRNA(1.00±0.00、7.18±0.41、12.88±0.45、32.84±1.61)표체균증강,차이유통계학의의(F=77.55,P＜0.05;F=379.0,P＜0.05).TGF-β2능유도HLEC전분화,농도의뢰성지촉진α-SMA단백(0.48 ±0.01、0.78 ±0.04、0.69±0.04)화mRNA(1.00 ±0.00、2.30±0.22、3.10±0.21、3.86 ±0.10)적표체,차이유통계학의의(F =62.73,P＜0.05;F=80.22,P＜0.05).불동농도(0.0、0.1、1.0、10.0 μg/L)TGF-β2종기인화단백수평촉진HLEC표체COL-Ⅰ화Fn,COL-Ⅰ mRNA적상대치분별위1.00 ±0.00、5.52±0.96、18.31 ±1.20、82.51 ±1.45,Fn mRNA적상대치분별위1.00 ±0.00、2.36±0.25、3.27 ±0.24、4.25±0.24；COL-Ⅰ단백표체상대치분별위0.78 ±0.05、1.15±0.11、2.16±0.14,Fn단백표체상대치위0.64±0.01、0.95 ±0.02、1.23 ±0.14,기작용정농도의뢰성(F=1880.52,105.30,P＜0.05;F=64.44,51.81,P＜0.05).결론 TGF-β2가이유도HLEC표체CTGF,촉진HLEC전분화급세포외기질적합성.
Objective To explore the effects of transforming growth factor-β2 (TGF-β2) on the transdifferentiation,extracellular matrix synthesis and connective tissue growth factor (CTGF) expression in human lens epithelium cells (HLEC) in vitro.Methods HLEC were incubated with different concentrations of TGF-β2(0.0,0.1,1.0 and 10.0 μg/L) for 24 h in vitro.The morphological changes of HLEC were observed under inverted phase-contrast microscope.The expression of CTGF and α-smooth muscle actin (α-SMA,as a landmark protein of epithelial mesenchymal transition) in HLEC was measured by immumofluorescence method.Real-time PCR and Western blot were used to evaluate the expression of CTGF,α-SMA,fibronectin (Fn) and COL-Ⅰ (as the major components of extracellular matrix) after stimulating by TGF-β2.Results Normal HLEC presented polygonal shape and were anchorage-dependent.After incubated with different concentrations of TGF-β2 for 24 h,the morphology of polygonal HLEC was changed into fibroblast-like shape and changed from monolayer and to multilayer cells,and the intercellular space became bigger.CTGF and α-SMA were expressed in the cytoplasm after induction of TGF-β2.Expression of CTGF in HLEC was increased with increasing concentrations of TGF-β2 (CTGF protein expression:0.53 ±0.03,0.73 ±0.01,0.65 ±0.03 in cells cultured with 0.1,1.0 and 10 μg/L TGF-β2,respectively; CTGF gene induction:1.00 ± 0.00,7.18 ± 0.41,12.88 ± 0.45,32.84 ± 1.61 in cells cultured with 0.0,0.1,1.0 and 10.0 μg/L TGF-β2,respectively) (F =77.55,P ＜ 0.05 ; F =379.0,P ＜ 0.05).TGF-β2 could induce HLEC transdifferentiation and accelerate.α-SMA expression was increased by TGF-β2 dose-dependently (protein expression:0.48 ± 0.01,0.78 ± 0.04,0.69 ± 0.04; gene induction:1.00±0.00,2.30±0.22,3.1 ±0.21,3.86±0.10)(F=62.73,P＜0.05; F=80.22,P＜0.05).TGFβ2 also promoted expression of Fn and COL-Ⅰ in a dose-dependent manner (COL-Ⅰ gene induction:1.00 ±0.00,5.52 ± 0.96,18.31 ± 1.2,82.51 ± 1.45 ; COL-Ⅰ protein expression:0.78 ± 0.05,1.15 ± 0.11,2.16±0.14;Fn gene induction:1.00 ±0.00,2.36 ±0.25,3.27 ±0.24,4.25 ±0.24; Fn protein expression:0.64 ±0.01,0.95 ±0.02,1.23 ±0.14) (F =1881.52,105.30,P ＜0.05 ;F =64.44,51.81,P ＜ 0.05).Conclusion TGF-β2 induces the expression of CTGF by HLEC,promotes transdifferentiation of and extracellular matrix synthesis by HLEC.
