【作者】 王晶；

【导师】 樊廷俊；

【作者基本信息】 中国海洋大学 ， 细胞生物学， 2010， 博士

【摘要】 角膜内皮细胞是存在于角膜靠近于房水面的六角形镶嵌排列的单层细胞,对维持角膜的透明有着关键作用。成年人角膜内皮细胞(HCEC)缺乏增殖能力,局部细胞受到损伤后,只能通过邻近细胞体积的增大和移行来填补缺损区,所以六角形HCEC的数量逐年减少,角膜透明度逐渐降低。现有研究结果表明,HCEC停留在细胞周期的早G1期,仍然保持有增殖的潜能。但关于HCEC停留在G1期的具体的细胞与分子机制,至今仍不清楚。转化生长因子β(TGF-β)属于TGF-β超家族的成员,具有调控细胞的增殖、分化、凋亡及组织的损伤后修复等作用。TGF-β2是存在于人房水中的主要的TGF-β类型,可促进小鼠角膜上皮层和基质层的正常发育及内皮层的形成,抑制体外培养的大鼠、兔和牛角膜内皮细胞的增殖、调控牛角膜内皮细胞外基质蛋白的合成以及角膜内皮细胞的损伤后修复。有关TGF-β2对HCEC增殖活性和形态的影响及相关的分子机理,至今还未见到相关的报道。为了探明TGF-β2对HCEC增殖活性的影响及相关的细胞和分子机理,本文利用体外培养的人角膜内皮细胞系,使用不同浓度的TGF-β2在含10%FBS的DMEM/F12的培养条件下处理HCEC,发现1-15ng/mL的TGF-β2对HCEC的增殖均有抑制作用,在1～9ng/mL的浓度范围内,TGF-β2对HCEC增殖的抑制作用具有浓度依赖性,其中9ng/mL为TGF-β2对HCEC增殖的峰值抑制浓度；9ng/mL的TGF-β2处理HCEC的细胞计数结果显示,TGF-β2处理12h即可抑制HCEC的增殖,抑制作用具有时间依赖性；流式细胞仪检测结果显示,9ng/mL TGF-β2处理后,处于G1/G0期HCE细胞的数量显著增加(P<0.05),并具有时间依赖性。荧光定量PCR结果显示,9ng/mLTGF-β2处理24 h后,HCEC对p27kip1的表达量显著增加,48 h后P27kip1和p21cip1的表达量均显著增加,也具有时间依赖性。以上结果说明,TGF-β2对HCEC具有显著的增殖抑制作用,并具有浓度和时间依赖性,其抑制作用机理很可能是通过先后诱导p27kip1和p21cip1表达量的增加进而将细胞拘留在G1/G0期来实现的。为了探明TGF-β2对HCEC形态的影响及相关的分子机理,本文首先采用1～15ng/mL的TGF-β2在含10%FBS的DMEM/F12的培养条件下处理HCEC,光学显微镜下观察的结果显示,3～15ng/mL的TGF-β2处理72h可明显增加细胞的贴瓶面积,在3-9 ng/mL的浓度范围内,TGF-β2对HCEC形态改变具有浓度依赖性,其中9ng/mL为TGF-β2对HCEC形态的峰值改变浓度；9ng/mL的TGF-β2处理HCEC的光镜观察结果显示,TGF-β2处理HCEC 24h即可增加单个细胞的铺展面积,促进细胞由上皮细胞样变为成纤维细胞样,形态的改变具有时间依赖性；细胞贴附实验表明,9ng/mL TGF-β2处理24h-72h均可明显的增强HCEC贴附能力(P<0.05)。荧光双染色结果显示,9ng/mL TGF-β2处理24h,可增加HCEC胞质中F-肌动蛋白丝和微管的长度,降低细胞中心体区域的荧光强度；处理72h则明显增加HCEC胞质中微丝束的数量和长度,并促进微管遍布于整个胞质空间；HCECⅠ型和Ⅳ型胶原的免疫荧光染色的结果显示,9ng/mL TGF-β2处理72h,可明显增加HCEC单个细胞胞外Ⅳ型胶原的铺展面积,降低其荧光强度；Western blot结果显示,9ng/mL TGF-β2处理24h和72h对HCEC I型胶原和Ⅳ型胶原的表达量均没有显著的影响。以上结果说明,TGF-β2通过增加平行于细胞长轴方向的F-肌动蛋白丝的数量、延长F-肌动蛋白丝和微管的长度以及增加Ⅳ型胶原的铺展面积,从而促进HCEC由上皮细胞样向成纤维细胞样的转变、增大单个细胞的铺展面积和增强细胞的贴附能力。本文研究了TGF-β2对体外培养的人角膜内皮细胞的增殖活性和形态影响作用,旨在探明TGF-β2在人角膜内皮细胞的增殖和形态改变中的作用及其相关的细胞和分子机理,为深入研究人角膜内皮细胞增殖调控的机理提供重要的理论支持,对了解TGF-β2在角膜内皮细胞的损伤修复过程中的作用具有重要的理论意义。更多还原

【Abstract】 Corneal endothelial cells are single layer of cornea near the aqueous humor, which is the key of keeping cornea transparency and normal thickness. Adult human corneal endothelial cell don’t normally replicate in vivo at a rate sufficient to replace dead and injured cells. And in vivo repair of the monolayer occurs mainly by cell enlargement and migration in response to cell loss. So the number of hexagonal cells and the transparency of cornea are reduced year by year. It is proved that the cell cycle status of HCEC is early G1 phase and the cells still have proliferation potential. But there is no idea about the reason now.Transforming growth factor-β(TGF-β) is belong to the TGF-βfamily, and regulates cell proliferation、differentiation、apoptosis and tissue repair after injury. TGF-β2 is present in the aqueous humor of the major human TGF-βtype, promotes mouse normal development of corneal epithelium and stroma and the formation of corneal endothelium,inhibits the proliferation of cultured rat, rabbit and bovine corneal endothelial cells, and regulates the extracellular matrix protein synthesis of bovine corneal endothelial cells and wound repair of corneal endothelium. And the molecular mechanism of TGF-β2 on HCEC proliferation and morphology has not yet been reported.In order to clarify cellular and molecular mechanisms of TGF-β2 on HCEC proliferation activity, this paper using different concentrations of TGF-β2 to treat HCEC, which is non-transduced human corneal endothelial cells belong to our lab, in DMEM/F12 containing 10% FBS.The results showed that 1～15 ng/mL of TGF-β2 had an inhibitory effect on proliferation of HCEC, and the inhibitory effect on proliferation was dose-dependent within 1～9ng/mL concentration range. The result of cell counting showed that the proliferation of HCEC was inhibited after 12h treatment by 9ng/mL TGF-β2 and the inhibition effect depended on the treated time. Flow cytometry:the proportion of HCEC G0/G1 phase cells was significantly increased after treatment by TGF-β2 for 24h-72h (P<0.05).Real-time PCR:Relative expression of G1 cell cycle kinase inhibitor P27kip1 increased after treatment by 9ng/mL TGF-β2 for 24 hours; and relative expression of P27kip1 and P21cip1 all increased after 48 hours and was time-dependent.These results suggest that, TGF-β2 significantly inhibited the proliferation of HCEC in a dose-and time-dependent manner. And the inhibition mechanism is likely to increase the expression of p27kip1 and p21cip1 one after another and arrest the cell at G1/G0 phase.In order to clarify the molecular mechanism of TGF-β2-on HCEC morphology, this paper used 1～15 ng/mL TGF-β2 treat HCEC in DMEM/F12 containing 10% FBS. The result showed that 3～15ng/mL of TGF-β2 could significantly increase attachment area of HCEC to the flask and promoted cells more fibroblast-like. Within 3-9ng/mL concentration range, the changes of HCEC shape was concentration-dependent. And was the the peak concentration of TGF-β2 for changing HCE cell shape.9ng/mL TGF-β2 could increase the attachment area of HCEC to the flask and promote cells more fibroblast-like in time-dependent manner. Immunofluorescence for F-actin and microtubules:the length of F-actin and microtubules was increased and fluorescence intensity centrosome area around the nuclear was decreased after treatment by TGF-P2 for 24 hours. And after 72 hours the number and length of microfilaments and microtubules were significantly increased and microtubules spread over the cytoplasmic space.The adhesion ability was enhanced by treatment by TGF-β2 for 24 or 72hours (P<0.05).The result of Immunofluorescence for collagen type I and collagen typeⅣshowed that 9ng/mL TGF-β2 could increase the spreading area of collagen typeⅣat the extracellular bottom of HCEC and decrease its fluorescence intensity. Western blot:Treatment by TGF-β2 for 24h or 72h had no significantly effect on the expression of collagen typeⅠand collagen typeⅣ.These results showed that TGF-β2 promoted HCEC more fibroblast-like, increased the attachment area of HCEC to the flask and enhanced adhering ability by increasing the number of F-actin、the length of F-actin and microtubule and the spreading area of collagen typeⅣbelow cell’s bottom.This is the first time to study the effect of TGF-β2 on human corneal endothelial cell proliferation and morphology in vitro. The Objective was to clarify the effect and mechanism of TGF-β2 on human corneal endothelial cell proliferation and cell morphology, which provides important theoretical guidance value to study the mechanism of human corneal endothelial cell proliferation regulation,and has important theoretical significance for understanding the function of TGF-β2 on the repair of corneal endothelium after injury.更多还原