ABSTRACT
Stroma is the major part of cornea.The factors maintain corneal stroma transparency include the ultrastructural anatomy and physiology of the cornea and its cellular and extracellular components.During corneal development,neural crest cells and keratoblasts differentiate into keratocytes,which synthesize high levels of collagen and proteoglycans.The production and assembly of collagen fibers need a series of intracellular and extracellular components.The assembled collagen fibers deposite in ECM,assemble into high-order structures to synthesize small fiber bundles,and then form a larger structure in a particular tissue.Corneal wound healing is completed in highly coordinate by various cells and cytokines in time and space.The keratocytes phenotype changes and the remodeling of corneal stroma ECM are two important factors in this process.Corneal refractive surgery are carried out in the worldwide,although most corneal refractive surgery are successful,But the stromal opacity formed during the remodeling of corneal stroma is the most important cause of visual impairment and visual quality decline after refractive surgery.Well understanding the course of stroma remodeling after corneal injury,is meaningful for the exploration of decreasing corneal opacity formation.In this paper,the corneal stroma structure and its synthesis and assembly,the pathophysiological process of stroma remodeling in corneal wound healing and after corneal refractive surgery were reviewed.
ABSTRACT
Background Transforming growth factor-β1(TGF-β1) plays an important role in corneal woundhealing.The effects of TGF-β1 on the synthesis of extra cellular matrix (ECM) vary upon different concentrations.Previous studies focused on the effects of high concentration of TGF-β1 on keratocytes under the two-dimensional culture condition,and the effect of low concentration of TGF-β1 on the synthesis of ECM in keratocytes remains unclear.Objective This study was to investigate the growth of Pellet,a three-dimensional model of corneal stroma cells in vitro,and its ECM synthesis under a low concentration of TGF-β1.Methods Bovine corneal stromal cells were isolated from fresh bovine eyeballs by two-step digestion by collagenase and cultured using DMEM/F12 medium with 10％ fetal bovine serum (FBS).Pellets derived fresh bovine keratocytes with culture medium containing 0.25 ng/ml TGF-β1 +5％ FBS and 0.50 ng/ml TGF-β1 +5％ FBS were established,respectively.The morphology of Pellets was observed under the natural light at 48 hours,1 week,2 weeks and 3 weeks after culture.In 3 weeks after culture,the cell structures was observed by hematoxylin-eosin staining,and Calcein-AM/propidium (Calcein-AM/PI) staining was used to assay the cell viability.Real-time flurorescence quantitative PCR and immunofluorescence technology were applied to analyze the expressions of α-smooth muscle actin (α-SMA),fibronectin (FN),type Ⅰ collagen (Col Ⅰ) and type Ⅲ collagen (Col Ⅲ) mRNA and proteins.RT-PCR was employed to detect the expressions of lumican (LUM) mRNA and keratocan (KERA) mRNA in the cells.Results Cells in Pellet clustered throughout the culture duration.Hematoxylin-eosin staining showed the mass red-dyed collagen fibers in both 0.25 ng/ml TGF-β1 +5％ FBS group and 0.50 ng/ml TGF-β1 +5％ FBS group,and most cells possessed complete structures.The death rate of the cells was (33.60±1.65)％ in the 0.25 ng/ml TGF-β1 +5％ FBS group and (30.90±0.78) ％ in the 0.50 ng/ml TGF-β1 +5％ FBS group,showing an insignificant difference between them (t =0.144,P=0.887).The expressions of α-SMA,FN and Col Ⅲ proteins in 0.25 ng/ml TGF-β1 +5％ FBS group were lower than those in the 0.50 ng/ml TGF-β1 + 5 ％ FBS group (tα-SMA =4.622,P =0.010;tFN =2.973,P =0.040;tCol Ⅲ =7.845,P＜0.001),but the expression of Col Ⅰ in 0.25 ng/ml TGF-β1 +5％ FBS group was higher than that in 0.50 ng/ml TGF-β1+5％ FBS group (tColⅠ =4.022,P=0.016).The ratio of Col Ⅲ/Col Ⅰ in 0.25 ng/ml TGF-β1+5％ FBS group was lower than that in the 0.50 ng/ml TGF-β1 +5％ FBS group in both mRNA and protein level (tmRNA =-3.039,P =0.038;tprotein =3.215,P =0.032).The expression of LUM mRNA and KERA mRNA were detected in Pellet at different time points.The expression of LUM mRNA in 0.25 ng/ml TGF-β1 +5％ FBS group increased over time.While in 0.50 ng/ml TGF-β1 +5％ FBS group,the expression of LUM mRNA peaked at 1 week but declined at 2 weeks.The expression of KERA mRNA in two groups were all peaked at 1 week but declined at 2 weeks.Conclusions Low-dose TGF-β1 in Pellet can maintain the normal growth of keratocytes and synthesize ECM.The expression of ECM tends to the normal condition after reducing the concentration of TGF-β1,implying a scarless expression.