Effect Of Lumican Expression On The Growth Of Sclera Fibroblasts In Rats And Its Mechanism

Reserch background and significance:The causes and mechanisms of the devolepment of human myopia are very complex and unclear.Sclera is generally considered as the main target tissue for myopic occurence.Scleral cells are mainly composed of scleral fibroblast(SF)and extracellular matrix(ECM).SF secretes the scleral collagen fibers that form the main part of the dense connective tissue of the sclera.During the process of myopia,obvious changes of morphology of ECM,especially collagen fiber may arise,causing scleral remodelling(SR),which changes the biomechanical properties of the scleral,reducing anti-pressure ability of the scleral,and making it easier to extend and deformat.At present,there have been a lot of relevant studies on the causes and specific mechanisms of scleral remodelling,but the harvest is still limited.As the Matrix Metalloproteinases(MMPs)and the tissue matrix metalloproteinase inhibitors(TIMPs)play important roles in ECM degradation,related studies have also been involved in the upstream cell pathway that causes the imbalance of MMPs and TIMPs secretion,but the specific mechanism is still unknown.Lumican,a member of the small leucine-rich proteoglycan family(SLRPs),is coded by the Lumican gene(LUM).Lumican protein is a component of scleral ECM of human eyes,and plays an important role in the regulation of scleral ECM,especially the synthesis and degradation of collagen.Thus it can be seen,Lumican,MMPs and TIMPs are involved in the myopic scleral ECM remodelling process.Therefore,to explore their expression and relationship in scleral tissue of experimental myopia as well as further study their role in SF growth is a worthy direction for understanding and elucidating the mechanism of myopic ECM degradation and even scleral remodeling.At present,there are few researches in this field at home and abroad.Based on this,two experiments were designed.In experiment 1,the expression changes of Lumican,MMP-2,MMP-14 and TIMP-2 in the scleral tissues of experimental myopia rats were detected.In experiment 2,the scleral SF of rats was cultured in vitro to construct the Lumican overexpression and interference vector,and further transfected with the SF separated from the primary generation.After successful transfection,the effects of different Lumican expression patterns on SF proliferation and apoptosis were detected,and the difference of expressions of Lumican,MMP-2,MMP-14 and TIMP-2 in corresponding groups were detected to explore the possible mechanism of Lumican's effect on SF growth.Part ?:Expression of Lumican,MMP-14,MMP-2 and TIMP-2 in sclera of form-deprivation myopia(FDM)rats Purpose:An experimental animal model of myopia in rats was established by using the method of palpebral suture for form deprivation.Observe the difference of Lumican,MMP-2,MMP-14 and TIMP-2 expression in scleral tissues of rats with form-deprivation myopia and normal controls.Methods:Twenty SD male rats aged 21 days were utilized.The right eye was selected as the experimental eye: the suture eyelid was performed for form deprivation for 8weeks.The left eye is regarded as a normal control eye.After 8 weeks of form deprivation,sixteen rats could pass muster later experiments.The model eye and the control eye were dilated respectively,and the diopter was detected by streak retinoscope.After the rats were killed,the eye axis was measured with vernier caliper immediately after the eyeball was removed.The morphological changes of scleral tissues in model and control eyes were observed under optical microscope.The ultrastructural changes of scleral collagen fibrils were observed by transmission electron microscopy.The relative protein expressions of Lumican,MMP-2,MMP-14 and TIMP-2 in two groups were detected by immunohistochemistry staining.The m RNA expressions of Lumican,MMP-2,MMP-14 and TIMP-2 in the two groups were detected by test methord of fluorescence quantitative PCR.Western blot exam was used to detect the protein expression of Lumican solely.Results:1.Compared with the control eye group,the model eye group developed significant high myopia,even super high myopia,with significantly increased negative refactive power(P<0.01,repectively),significantly increased ocular axis length(P<0.05,repectively)2.Under optical microscope,the sclera structure of normal control eyes was regular with clear layers.In the model group,the sclera was obviously thinned,the collagen fibers were sparse,the gap was enlarged and the fibers were broken.Compared with the control eye,under electron microscope,in the model eyes,collagen fibrils in the sclera were loosely arranged,malformed large and small fibrils were observed,and the fiber gap was obviously enlarged.The structure of fibreboard layer was damaged and the interlacing state was disordered.Irregular fiber distribution could be observed and it could be found that part of collagen fibrils have cavitation in cross section.3.Compared with the control eye group,m RNA expressions level of Lumican and TIMP-2 m RNA in the sclera tissues of the model eye group rose significantly,while expressions level of MMP-2 and MMP-14 m RNA declined significantly(P<0.05,respectively).4.Compared with the control eye group,the protein expressions of Lumican and TIMP-2 in the sclera tissues of the model eye group were significantly ascending,while the protein expressions of MMP-2 and MMP-14 were significantly descending(P<0.05,respectively).Conclusion:1.Obvious changes of scleral remodeling occurred in myopic rats with form-deprivation.2.Experimental myopia induced by form-deprivation in rats resulted in increased protein expression levels of Lumican and TIMP-2 and decreased protein expression levels of MMP-2 and MMP-14 in experimental eye sclera tissues.Part ?: Effects of Lumican gene(LUM)overexpression on the growth and expression of MMP-14,MMP-2 and TIMP-2 in rat sclera fibroblasts cultured in vitro Objective:To study the effects of different Lumican gene(LUM)expressions on the proliferation and apoptosis of rat fibroblasts cultured in vitro,and to detect the changes of corresponding m MP-14,MMP-2 and TIMP2 protein contents level,and to explore the possible mechanism of action.Methods:1.Primary rat scleral fibroblasts were isolated and identified,Lumican overexpression and interference vector were constructed,and scleral fibroblasts were transfected.Test methord of Fluorescence quantitative PCR and Western blot were used to verify the effect of overexpression.2.Experimental grouping: 1)Normal group(Control);2)Overexpression of no-load group(NC);3)Lumican overexpression group;4)Interference no-load group(si RNA-NC);5)Lumican interference(Lumican-si RNA).CCK8 assay was used to test cell viability and flow cytometry was used to test apoptosis rate in each group.Western blotting was used to inspection the expression of MMP-2,MMP-14 and TIMP-2 proteins in each group.Results:1.Lumican overexpression and interference vector were successfully constructed and transfected into cultured rat scleral fibroblasts.The experiment result of Lumican overexpression was effctive.2.Compared with the control group,cell proliferation of Lumican overexpression group were significantly slower and apoptosis accelerated(P<0.05,respectively).3.Compared with the control group,MMP-2 and MMP-14 protein expressions in the Lumican overexpression group were significantly decreased,and TIMP-2protein expression was significantly increased.In Lumican interference group,MMP-2 and MMP-14 protein expressions were significantly upregulated,while TIMP-2 protein expression was significantly downregulated(P <0.05,respectively).ConclusionOverexpression of Lumican in normal scleral fibroblasts in rats could induce accelerated apoptosis and reduced cell survival rate,and its mechanism may be related to down-regulation of m MP-2 and MMP-14 expression levels and up-regulation of TIMP-2 expression levels.