The Effect And Mechanism Of Transforming Growth Factor-β2 On Human Corneal Endothelial Cell Proliferation And Morphology In Vitro

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.