| Research background:Cornea is located in the front of the eye of a layer of transparent film,about the fiber membrane before the 1/6,from the back to see the cornea was round,the normal cornea is no blood vessels,blood vessels stop at the limbus,the formation of vascular network,this The neovascular network is called corneal neovascularization(CNV).Neovascularization is a sign of corneal inflammatory inflammation,the occurrence and development of neovascularization depends on the nature of inflammatory stimuli,once the blood vessels from the normal vascular invasion into the cornea,is the corneal pannus.Corneal neovascularization is a compensatory response to various pathogenic factors in the body of the cornea.With its elimination of infection,wound healing,inhibition of immune-mediated corneal dissolution has a certain effect,but its structure and function is not perfect,easy The occurrence of plasma leakage,resulting in corneal edema,lipid deposition and stimulate the cornea scar,a serious impact on vision;also make the incidence of corneal graft rejection greatly increased serious harm to patients with vision,inhibition of corneal neovascularization has been the development of eye The problem.MiR296 is located at chromosome 20q13.32,in the whole body disease and part of the organ in vitro experiments,has been confirmed miR-296 by directly inhibiting the target gene FGF23 expression,can promote the role of neovascularization,some scholars have confirmed miR-296 molecules The role is related to neovascularization and is a better inhibitor of neovascularization.However,there are few reports on corneal neovascularization associated with miRNA-296.In the present study,This experiment will be through the two parts of the first part: through the establishment of mouse model of alkali burn after the clear expression of mi R-296 in corneal neovascularization;the second part discusses to elucidate the molecular mechanisms involved in the regulation of miR-296 corneal neovascularization,provide new methods of treatment for neonatal vascular membrane caused by angle of various pathogenic factors Research content:BALB/ C mice 78,by the Nanchang University School of Medicine Experimental Animal Science Department,the weight of 35 ~ 45 g,all male;eyes are experimental eyes.A total of 78 mice were randomly divided into two experimental groups and one control group.The experimental group A: clopidogrel suspension eye drops and levofloxacin eye drops;experimental group B: tacrolimus eye drops Fluid + levofloxacin eye drops;control group C: levofloxacin eye drops 3 times a day as a negative control,a total of 21 days observation.All experimental animals were started 1 day after modeling,given levofloxacin eye drops 4 times a day,evenin three days.Part one: Establishment of corneal neovascularization model and detection of miRNA296 expression in vitro:1.1 the length of corneal neovascularization was observed and measured by slit lamp microscope to calculate the area of neovascularization and record the number of neovascularization.1.2 immunohistochemical staining was used to detect the expression of VEFE in corneal neovascularization after alkali burn at different time points1.3 The expression of miRNA-296 in corneal neovascularization was detected by qRT-PCR in BALB / C mice at different time points,and the dynamic changes of corneal neovascular miRNA 296 after alkaline burn were observed.Result:(1)RT-PCR was used to detect mi RNA296 in corneal tissue.The relative expression of each experimental group and control group at each time point after modeling was lower than that of experimental group B and control group C(P <0.05)(2),W-B detection of corneal tissue miRNA296 target gene FGF23 expression.The expression of FGF32 protein in the experimental group and the control group at each time point was lower than that in the experimental group B and the control group(P <0.05),and the difference was statistically significant(P <0.05)(0.245 ± 0.024)was significantly higher than that in control group(1.136 ± 0.126).The expression of miR-296 in corneal neovascularization was significantly higher than that in control group(1.136 ± 0.126))Was significantly down(P <0.01).(4).MiRNA 296 in the chip technology to detect FGF23 mRNA expression levels decreased,the protein level was significantly decreased.At the same time,the mRNA and protein expression levels of VEFE were significantly up-regulated,while the mRNA and protein expression levels of FGF23 were significantly decreased(P <0.05)(5)The target gene predicted by miRNA was correlated with FGF23,VEGF and IL-6 angiogenesis signaling pathway by microarray.Conclusion:1)clopidogrel suspension eye drops can effectively inhibit the proliferation of corneal neovascularization in alkali burned mice,and can reduce the corneal neovascularization.2)tacrolimus eye drops can effectively inhibit the proliferation of corneal neovascularization in mice after alkali burn,but the effect is not effective with clopidol suspension eye drops to inhibit the proliferation of corneal neovascularization.3)The results of immunohistochemical staining showed that the number of inflammatory cells and the number of inflammatory cells in the corneal neovascular tissue of experimental group A and group B were decreased compared with those in control group C at 7 days after corneal alkali burn.Rat corneal neovascularization,while hormones and immunosuppressive agents can inhibit the expression of VEGF4)the relative expression amount of miRNA296 in the RT-PCR corneal tissue at each time point after modeling,the A group in the treatment group was lower than that in the treatment group(P<0.05)and the control group,the difference was statistically significant(B)Part two: the molecular mechanism of miR-296 involved in the regulation ofcorneal neovascularization Method:2.1 Western blot was used to detect the expression of FGF32 protein in BALB / C mice at different time points after alkali burn.The expression of miR-296 target gene FGF32 in neonatal mice after alkaline burn at different time points Protein expression changes.2.2 The target genes of miRNA296 were predicted by microarray technology,and the signaling pathways of FGF23,VEGF and IL-6 corneal neovascularization were investigated.Result:(1)W-B detected the expression of FGF32 protein in corneal tissue at different time points after miRNA296,the treatment group A group was lower than the treatment group B and control group,the difference was statistically significant(P<0.05)(2)the model of corneal neovascularization was established,and the expression of miR-296 in corneal neovascularization of rats with alkali burn(0.145 +.0.013)was significantly higher than that of normal cornea(+ + 1.015 +)(P < 0.01).(3)the expression level of FGF23 mRNA was decreased and the protein level of was decreased significantly in the chip technology of miRNA.At the same time,the mRNA and protein expression levels of VEFE were significantly increased and the mRNA and protein expression levels of FGF23 were significantly decreased(P<0.05)(4)detection of the target gene by microarray technique miRNA is related to FGF23,VEGF,and IL-6 signaling pathway Conclusion:1)qRT-PCR results showed that the expression of miR-296 in corneal neovascularization was lower than that in normal corneal tissue(P <0.05)at all time points after corneal alkali burn,and miR-296 in group A(0.402387 ± 0.126035),the most obvious at day 10(0.432532 ± 0.138526),indicating that the corneal neovascularization in the cornea of the mice was the same as that of the miR(P <0.05),and the expression of miR-296 expression,and alkali burns can stimulate the gradual reduction of miR-296 expression levels.2)Western-blot results showed that the expression of FGF23 protein in the corneal neovascularization of the experimental group was significantly decreased at 1d,3d and 7d after corneal neovascularization at all time points after corneal alkali burn.The corneal alkali burn MiR-296 expression in corneal neovascularization inhibits protein expression of its target gene FGF23.3)Western blot analysis miR-296 can up regulate the VEGF-VEGFR signaling pathway in corneal endothelial cells and down regulate the FGF23 signaling pathway4)miR-296 target gene bioinformatics prediction screening differences in gene analysis We used the screening of alkali burns in the process of corneal neovascularization induced screening process of 24 genes,including FGF23,VEGF,bFGF,IL6,etc 9.Inflammatory factors are involved in the expression of corneal neovascularization... |
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