| Objective:To establish a three-dimensional culture system simulating the spatial dynamic distribution of transforming growth factor-?(TGF-?)during corneal matrix healing in vitro.Methods:Corneal stromal cells were isolated from fresh bovine eyes and cultured in DMEM/F12 culture medium containing volume fraction of 10%FBS.Then a three-dimensional culture model of Pellets was established for culture.Pellets were divided into dialysis tubes group and non-dialysis tubes group.After 48h of culture in the upper and lower compartments of Transwell chamber system,the fluid in the upper chamber was changed to 0.5 ?g·L-1TGF-?1+0.25?g·L-1TGF-(32+volume fraction of 10%FBS and volume fraction of 10%FBS in the lower chambers.The morphological changes of Pellets,culture model were observed 72 h after culture.Real-time PCR was used to detect the relative expressions of a-smooth muscle actin(?-SMA),fibronectin(FN),type ? collagen(Col ?)and type ? collagen(Col ?)mRNA in each group.Results:After 72 hours of culture,Pellets grew into clusters.In the upper and lower chambers of dialysis tubes group,the expression levels of:a-SMA mRNA were 1.595 ±0.025 and 1.148±0.009;FN mRNA were 1.090±0.011 and 0.844±0.015;Col I mRNA were 1.445±0.035 and 1.165±0.008;Col ? mRNA were 1.726±0.031 and 1.314±0.020;and the ratio of Col ?/Col ? were 1.126±0.019 and 0.957 ±0.013.The differences between the upper and lower chambers were statistically significant(all P=0.000).In the upper and lower chambers of non-dialysis tubes group,the expression levels of:?-SMA mRNA were 1.363±0.018 and 1.360±0.002;FN mRNA were 0.946±0.017 and 0.952+0.012;Col I mRNA expression were 1.261±0.011 and 1.258±0.029;Col ?mRNA were 1.459±0.027 and 1.462±0.033;and the ratio of Col ?/Col I were 1.157±0.029 and 1.163±0.090.There was no significant statistical difference between upper and lower chambers of non-dialysis tubes group(all P>0.05).The relative expressions of a-SMA,FN,Col ? and Col ? in the upper chamber of the non-dialysis tubes group were significantly different from those of the upper and lower chamber of the dialysis tubes group(all P<0.05).The relative expressions of a-SMA,FN,Col ? and Col ? in the lower chamber of the non-dialysis tubes group were also significantly different from those of the upper and lower chamber of the dialysis tubes group(all P<0.05).Conclusion:The combination of the Transwell chamber system and dialysis tubes is able to establish a three-dimensional culture system which can simulate the spatial dynamic distribution of TGF-? in vitro.Background:Transforming growth factor(TGF)? participates in and mediates the process of corneal injury repair,induces fibrosis of corneal extra cellular matrix(ECM),causes deposition of pathological ECM,and leads to formation of scar.Present studies wound healing in vitro generally based on two-dimensional culture,but TGF-? in corneal wound healing actually has the dynamic changes of the space and time,the expression of TGF-?1 and TGF-?2 in the damaged area varied with time,TGF-?1 can be spread to undamage area in the middle of the damage period,promotes the proliferation and the migration from surrounding area to the damaged area to promote wound healing.However,there are few reports on the correlation of the damaged and undamaged area and growth factors.In our previous work,we established an ECM fibrosis model of corneal stromal cells in vitro.In this study,we intend to improve the three-dimensional culture system to study the effect of spatial and temporal changes of growth factors on corneal wound healing.Objective:To establish a three-dimensional culture system simulating the spatial dynamic distribution of TGF-? during corneal matrix healing in vitro.Methods:Bovine corneal stromal cells were isolated from fresh bovine eyeballs,then establish a three-dimensional culture model of Pellet that derived flesh bovine keratocytes in vitro for culture.Pellets were divided into three groups,cultured in centrifuge tube of 15ml and the upper and lower chambers of Transwell chamber system,were respectively the basic medium group(named the blank control group),the group simulating spatial and temporal distribution of TGF-? in damaged area cultured in upper compartments(named the upper chamber group),and the group simulating spatial and temporal distribution of TGF-(3 in undamaged area cultured in lower compartments(named the lower compartment group).The blank control group was continuously cultured in basic medium for 21d.After 48h of culture in Transwell chamber system,the fluid in the upper chamber was changed to 0.5 ?g-L-1 TGF-?1+0.25?g·L-1TGF-?2+basic medium and basic medium in the lower chambers.After 72h of culture,it was changed to 0.5?g·L-1 TGF-?10.5?g·L-1 TGF-?2+basic medium and basic medium;after 5d of culture,it was changed to 0.5?g-L-1 TGF-?1+1.0?g·L-1 TGF-?2+basic medium and basic medium;after 7d of culture,it was changed to 0.5?g·L-1 TGF-?1+0.5?g·L-1 TGF-?2+basic medium and 0.5pg·L-1 TGF-?1+basic medium;after 9d of culture,it was changed to 0.25?g·L-1 TGF-(32+basic medium and 0.25?g·L-1 TGF-?1+basic medium;after 14d of culture,it was changed to basic medium.The morphology of Pellets was observed under the natural light at 48h,72h,5d,7d,14d,21 d after culture.Calcein-AM/propidium(Calcein-AM/PI)staining was applied to assay the cell viability of Pellets.Real-time PCR was applied to detect the mRNA expressions of a-smooth muscle actin(a-SMA),fibronectin(Fn),type I collagen(Col I),type III collagen(Col III)mRNA,Keratocan(KERA)and Lumcan(LUM).Immunofluorescence(IF)was applied to detect the protein expression of a-SMA,FN,Col I and Col III at 48h,7d,14d after culture.Transmission electron microscope was used to detect the diameter,size and spatial arrangement of collagen fibers in Pellets.Results:Pellets in Transwell stayed cluster,were able to sustain long-term cultivation.Calcein-AM/PI staining results show that the mortality of cells in Pellets of three groups increased with time,but there are still plenty of living cells.Real-time PCR results show that expression levels of fibrosis markers a-SMA,FN,Col I mRNA and the ratio of Col III/Col I of upper chamber group were higher than the lower chamber group and blank control group,and there was no significant statistical difference between lower chamber and blank control group at 48h,72h,5d,7d after culture.The expression of a-SMA,FN,Col III mRNA and the ratio of Col III/Col I of upper chamber group were higher than the lower chamber group and blank control group at 14d and 21d after culture(all P<0.05).LUM and KERA mRNA could be detected at 48h,72h,5d,7d,14d,21d after culture in three groups.The expression of LUM and KERA mRNA of bland control group was higher than upper and lower chamber group(all P<0.05).IF results show that the protein expression of a-SMA,FN,Col I and Col III could be detected at 48h,7d,14d after culture,the protein expression of ?-SMA,FN,Col III mRNA and the ratio of Col III/Col I of upper chamber group were higher than the lower chamber group and blank control group at 7d and 14d after culture(all P<0.05),and there was no significant statistical difference between lower chamber and blank control group at 48h and 7d after culture.At 21d after culture,the observation results with transmission electron microscope show that the arrangement and diameter uniformity of collagenous fibers was orderly and uniform of blank control group,disorder and irregular of upper chamber group.The arrangement and diameter uniformity of collagen fibers of lower chamber group was neat,and the uniformity was close to the blank control group.Conclusion:The upper chamber of Transwell that TGF-?1 and TGF-?2 was added to can simulate the temporal and spatial changes of TGF-? in the damaged area during corneal matrix injury,and significant ECM fibrosis of Pellets in the upper chambers occurred and occurred earlier than the Pellet cultured in TGF-?1 only.The lower chambers that TGF-?1 was added to simulate the low expression of TGF-P in the undamaged area during corneal matrix injury,and mild fibrosis occurred in Pellets.Compared with the blank control group without TGF-?,ECM fibrosis was more significant in upper and lower chambers.The three-dimensional culture system combined with the spatial and temporal dynamic control of TGF-? in the upper and lower chambers can be used to simulate the dynamic distribution of TGF-? in the damaged area and undamaged area of corneal matrix wound healing in vivo,and can be used as a candidate model for three-dimensional culture of corneal matrix wound healing in vitro. |
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