摘要
●AIM:To explore netrin-1 functions on corneal epithelium in vitro and in vivo.●METHODS:In vitro the human corneal epithelial(HCE)cells were treated with serum free DMEM-F12 basic media containing 0,50,100,200,300,500,800,and 1000 ng/mL of netrin-1,respectively.The cells viability was detected by cell counting kit-8(CCK-8).The wound-healing assay was applied to assess the migration proficiency of HCE cells.Flow cytometry was used to analyze the cell-cycle distribution and apoptosis.In vivo,normal c57(6 wk)mice were demarcated with a trephine in the middle of the cornea to produce a 3-mm circular wound.Mice corneas were inflicted no epithelium with a 3-mm wound displayed,but remained the limbal epithelium intact.A blunt scalpel blade was used to remove the corneal epithelian cells,followed by topical netrin-1 application(200 ng/mL),and the group treated by PBS as control.The treated group was injected netrin-1 into the normal c57 mice inferior subconjunctival 4 h before trauma.Mouse corneal inflammation and neovascularization were observed under slit lamp microscope.The apoptosis of corneal cells was determined by TUNEL staining.●RESLUTS:A concentration of 200 ng/mL netrin-1 enhanced 25%of the HCE viability.The relative migration rates were 76.3%and 100%in control and netrin-1 treated group after cultured 72 h.Treated with netrin-1(200 ng/mL)decreased the apoptosis of HCE cells,as well as decreased their percentage from 19.3%±0.57%to 12.7%±0.42%of the total.The remaining wound area was 1.22 mm2 in control group but 0.22 mm2 in the netrin-1 treated group.Exogenous Netrin-1 inhibits apoptosis of corneal epithelial cells of c57 mice.TUNEL-positive cells at the epithelial layer of the corneas of the control and netrin-1 treated c57 mice at 24 h after wounding were 43.3%and 16.7%respectively.●CONCLUSION:Netrin-1 can reduce HCE apoptosis as well as promote its proliferation and migration.Topical application of netrin-1 promotes the injuryed cornea epithelial wound repair and inhibits apoptosis of corneal epithelial cells.These findings may offer potential therapies to repair the defects of corneal epithelial based on netrin-1.
● AIM: To explore netrin-1 functions on corneal epithelium in vitro and in vivo. ● METHODS: In vitro the human corneal epithelial(HCE) cells were treated with serum free DMEM-F12 basic media containing 0, 50, 100, 200, 300, 500, 800, and 1000 ng/mL of netrin-1, respectively. The cells viability was detected by cell counting kit-8(CCK-8). The wound-healing assay was applied to assess the migration proficiency of HCE cells. Flow cytometry was used to analyze the cell-cycle distribution and apoptosis. In vivo, normal c57(6 wk) mice were demarcated with a trephine in the middle of the cornea to produce a 3-mm circular wound. Mice corneas were inflicted no epithelium with a 3-mm wound displayed, but remained the limbal epithelium intact. A blunt scalpel blade was used to remove the corneal epithelian cells, followed by topical netrin-1 application(200 ng/mL), and the group treated by PBS as control. The treated group was injected netrin-1 into the normal c57 mice inferior subconjunctival 4 h before trauma. Mouse corneal inflammation and neovascularization were observed under slit lamp microscope. The apoptosis of corneal cells was determined by TUNEL staining. ● RESLUTS: A concentration of 200 ng/mL netrin-1 enhanced 25% of the HCE viability. The relative migration rates were 76.3% and 100% in control and netrin-1 treated group after cultured 72 h. Treated with netrin-1(200 ng/mL) decreased the apoptosis of HCE cells, as well as decreased their percentage from 19.3%±0.57% to 12.7%±0.42% of the total. The remaining wound area was 1.22 mm2 in control group but 0.22 mm2 in the netrin-1 treated group. Exogenous Netrin-1 inhibits apoptosis of corneal epithelial cells of c57 mice. TUNEL-positive cells at the epithelial layer of the corneas of the control and netrin-1 treated c57 mice at 24 h after wounding were 43.3% and 16.7% respectively. ● CONCLUSION: Netrin-1 can reduce HCE apoptosis as well as promote its proliferation and migration. Topical application of netrin-1 promotes the injuryed cornea epithelial wound repair and inhibits apoptosis of corneal epithelial cells. These findings may offer potential therapies to repair the defects of corneal epithelial based on netrin-1.
基金
Supported by the National Natural Science Foundation of China(No.81300729,No.81160118,No.81460092,No.81660158)
Natural Science Foundation of Fujian Province(No.2015J05170).
