| ObjectivePosterior capsular opacification(PCO)is one of the common complications after modern cataract surgery,and the epithelial-mesenchymal transition(EMT)of the residual lens epithelial cells(LECs)is the pathological basis of PCO.EMT is also a pivotal mechanism of malignant tumor.The up-regulation,down-regulation or structural change of Notch signaling has something to do with the occurrence of various malignant tumors,meanwhile the up-regulation of micro RNA34a(mir-34a)as an effective tumor suppressor gene can induce cell apoptosis and cell cycle arrest.This study intends to investigate the role of mi R-34a/Notch signaling pathway in the LECs-EMT phenotype acquisition in vivo animal models.Whereas lens regeneration is a continuation of PCO process;corneal epithelium and lens have the same tissue origin in early embryonic development;Notch signaling pathway blocker(DAPT)injected into the anterior chamber will have corresponding effects on all tissue structures of the anterior segment in vivo,etc.In view of the above reasons,this study also intends to observe the mechanism of mi R-34a/Notch signaling pathway in lens regeneration and corneal epithelial injury repair,as well as the effects of its changes on these two biological processes.MethodsPart Ⅰ: The PCO model of rat was constructed.The development of PCO in the normal group and the 1μmol or 5μmol DAPT injected into anterior chamber group of different doses were compared by anterior segment imaging and HE staining.RT-q PCR was used to detect the specific markers expression of posterior capsule tissue samples in each group,including receptors Notch1、Notch2,ligands Jag1、DLL1 and downstream gene Hes1 of Notch signaling pathway,Snail,Slug and Zinc finger e-box binding homeobox 1 of EMT transformation factor,alpha-smooth muscle actin,vimentine and nuclear proliferation marker Ki67 of mesenchymal cell and mi R-34a;Part Ⅱ:The lens regeneration model of rat was constructed.The morphologic changes of lens regeneration were observed by anterior segment imaging and HE staining.RT-q PCR was used to detect the specific markers expression of lens regeneration tissue samples,including receptors Notch1、Notch2,ligands Jag1 of Notch signaling pathway,crystallin,beaded fibrous structural protein1 and aquaporin of lens fiber,fibroblast growth factor 2 of lens growth induction factors and mi R-34a;Part Ⅲ:The corneal epithelial injury repair model of rat was constructed.The development of epithelial injury repair in the normal group and the 1μmol or 5μmol DAPT injected into subconjunctival group of different doses were compared by anterior segment imaging and HE staining.RT-q PCR was used to detect the specific markers expression of corneal tissue samples in each group,including receptors Notch1 、 Notch2,ligands Jag1 of Notch signaling pathway,Zona occludens-1,Keratin12 and nuclear proliferation marker Ki67 of corneal epithelial cell and mi R-34 a.Results1.Using PCO model,the expression of Notch signaling pathways began to rise at extracapsular lens extraction(ECLE)postoperative 3 days(d),peak at 7-14 d,returned to baseline at 21-28 d.The changes of EMT transformation factors and mesenchymal cell markers were highly consistent with Notch signal.The expression of mi R-34 a gradually decreased after ECLE,reached a low point at 7d,gradually increased at the 14 d and doubled at 28 d.After 5μmol DAPT injected into the anterior chamber,the development of EMT was inhibited as while as the Notch pathway.The inhibitory effect was more significant at 3-14 d.However,the expression of mi R-34 a was increased,and the difference was statistically significant compared with the normal group at 7-14d;2.Using lens regeneration model,the regenerated lens proliferated rapidly at ECLE postoperative 30 d,and basically reached the normal lens size at 60-90 d.However,the internal structure of the regenerative lens is partly cloudy,disordered and loose.During the lens regeneration,the expression of mi R-34 a was increased,which negatively regulated the Notch signaling pathway,decreased the related markers expression and promoted the proliferation of differentiated lens fibroblasts.But the expression of lens fibrous tissue related markers and lens growth induction factors was lower than normal,and the difference was statistically significant;3.Using corneal epithelial injury model,the expression of markers related to Notch signaling pathway gradually decreased after injury,reached the lowest point between 24 and 48 h,and then gradually recovered,and still did not rise to the normal expression level at 72 hours after injury.However,the expression of mi R-34 a gradually increased after injury,reached a peak around 24 h,and then gradually decreased.By the end of observation,the mi R-34 a expression had not returned to the normal undamaged state,and the change of mir-34 a expression was negatively correlated with the that of Notch signaling pathway.The expression of mi R-34 a increased and the expression of Notch signaling pathway decreased at the early stage of repair,which promoted epithelial cell proliferation and wound healing rapidly.At the middle and late stage of repair,the expression of mi R-34 a decreased and the expression of Notch increased,which inhibited the excessive proliferation of epithelial cells and promoted differentiation,thus promoting the recovery of corneal epithelial barrier function.5 μ mol DAPT subconjunctival injection effectively inhibited the Notch signaling pathway and promoted epithelial cell differentiation and the recovery of barrier function,but had no significant effect on proliferation.ConclusionsDuring the occurrence of PCO,the mi R-34a-Notch signaling pathway regulates the changes of LECs-EMT,and the inhibitory effect of 5μmol DAPT injection into the anterior chamber is more significant.Taking mi R-34a-Notch signaling pathway as a target may provide new ideas and directions for the treatment of PCO in the future.During the occurrence of lens regeneration,mi R-34 a expression was increased,which negatively regulated the Notch signaling pathway and promoted the proliferation of differentiated lens fibroblasts,but the function and structure of the regenerative lens were lower than normal.During the occurrence of corneal epithelial injury repair,the mi R-34a-Notch signaling pathway regulates the degree of proliferation and differentiation of epithelial cells at different time periods,and 5 μmol DAPT subconjunctival injection promoted the differentiation of epithelial cells and the recovery of barrier function in the middle and late repairing stage,but had no significant effect on proliferation. |
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