| Objective: To construct the human immunodeficiency virus(HIV)with expression of c-ski /GFP gene.HIV-c-Ski/GFP was used as the observation group,and HIV-GFP used as the control group,which was packaged as an infected virus particle(completed in our previous studies).The model of chronic intraocular pressure(IOP)was established in mice.After transfected HIV-c-Ski/GFP /HIV-GFP genes into the anterior chamber of mice,we observed the c-Ski gene expression in TM,and explored the effects of c-Ski gene on IOPin mice,extracellular matrix(ECM)of the trabecular meshwork and related possible mechanism,thus illustrated the mechanism of c-Ski gene IOP regulation.Methods: 60 adult health male C57BL/J6 mice randomly divided into 5 groups,experimental animal grouping specifically as follows: normal + HIV-GFP group(A),normal + HIV-c-Ski/GFP group(B),glaucoma group(C),glaucoma + HIV-GFP group(D),glaucoma + HIV-c-Ski/GFP group(E).(1)normal + HIV-GFP group(A): the mice were intracamerally injected 2μL HIV-GFP in the right eye,and the virus titer was 3.4 x 108TU/ml;(2)normal + HIV-c-Ski/GFP(B): the mice were intracamerally injected 2μL HIV-c-Ski/GFP in the right eye HIV-c-Ski/GFP,and the virus titer was same as HIV-GFP;(3)glaucoma group(C): the mice were intracamerally injected 10μm micriobeads PBS mixed suspension 2μL in the right eye,and the number of microbeads was about 7000.(4)glaucoma + HIV-GFP group(D): the mice were intracamerally injected 2μL HIV-GFP.1 week later,10μm microbeads PBS mixed suspension 2 ul was intracamerally injected into the same eye,and the number of microbeads was same as group C.(5)glaucoma + HIV-c-Ski/GFP group(E): the mice were intracamerally injected 2μL HIV-c-Ski/GFP.1week later,10μm microbeads PBS mixed suspension 2μL was intracamerally injected into the same eye,and the number of microbeads was same as group C.Before virus injection,IOP were measured as baseline using rebouond tonometry.After 10μm micriobeads PBS mixed suspension 2μL injected into the anterior chamber of mice,IOP in the right eye were measured every three days for four weeks using tonolab rebound tonomery.Then,animals were sacrificed on day 28,the right eyes were enucleated,hematoxylin-eosin staining was performed on trabecular meshwork(TM)cross-sections for observe the cornea,anterior chamber,TM,Schlemm trube,iris structure.TM cross-sections were used to immunofluorescence(IF)to detect ECM related protein(Fiberonectin(FN),Laminin(LN),β-catenin fluorescence intensity expression.Also,TM proteins were obtained by tissue homogenation,and western blot to detect such as transforming growth factor beta-2(TGF-β2),phosphorylation Smad2(p-Smad2)/Smad2,phosphorylation Smad3(p-Smad3)/Smad3,c-Ski,FN,LN,and β-catenin protein expression level.Results: 1.Before HIV intracameral injection and after10μm micriobeads PBS mixed suspension 2μL intracameral injection on day 3、day 7、day 14 and day 28,operation microscope was used to obverse operative eye,if eye was hyperaemia or not,cornea was transparent or not,anterior chamber had obvious inflammation or not,iris was bleeding/incarcerated or lens was transparent or not and glass body cavity was turbidity or not.2.IOP results: Before HIV tramsfection,baseline IOP in every group was no significant different(P > 0.05).After intracamerally injected 10μm micriobeads PBS mixed suspension 2μL in group C,D,E,IOP increased significantly(P > 0.05)compared to the group A and B at each time point,while IOP in group A and B had no significant increase in each time point(P > 0.05)compared to the baseline intraocular pressure.In the glaucomous model groups,IOP in group E was significantly decreased,compared with group C and D,moreover IOP peak value reduced(P < 0.05).3.The TM cross-sections of the optical microscope after HE staining examination showed: no obvious inflammation reaction and structure change were detected in cornea,anterior chamber,TM,iris in each group.4.Immunofluorescence(IF)staining and fluorescence microscope examination revealed: Compared with those in group A and B,FN,LN and β-catenin fluorescence intensity expression in group C,D and E were enhanced obviously,which were located at the around TM,JCT and Schlemm tube,while there was no obvious difference compared group C with group D.FN,LN and β-catenin fluorescence intensity expression in group E(with the c-Ski gene transfection)were significantly reduced,compared with group C and D;As for the expression of c-Ski,the fluorescence intensity expression were significantly enhanced in group B and E due to the c-Ski gene transfection,compared with group A,C and D.5.Western bolt revealed: FN,LN and β-catenin protein expression level in each glaucomous group were increased significantly,compared with group A and B(P < 0.05),however,there was no significant difference compared group C with group D(P > 0.05).FN,LN and β-catenin protein level was significantly reduced in group E(with the c-Ski gene transfection)compared with group C and D(P < 0.05);c-Ski protein expression was significantly increased in group B and E compared with group A、C and D(P > 0.05),and there was no difference in c-Ski protein expression between group A,C and D(P > 0.05).compared with group A and B TGF-β2 protein expression levels in glaucomous groups(group C,D and E)were significantly increased,compared with those in non-glaucomous groups(group A and B)(P < 0.05).Group E transfected with c-Ski gene had a reduced TGF-β2 expression and an increased p-Smad2/p-Smad3 expression compared with those in group C and D(P < 0.05)Smad2/Smad3 protein expression level had no difference in each group(P > 0.05).Conclusions: 1.HIV-c-Ski/GFP could be effectively transfected in the TM,JCT and Schlemm tube of mice,and no obvious inflammation had been seen in mice eyes.2.Mouse chronic IOP elevation model induced by intracameral injection of 10μm microbeads can lead to the change of ECM proteins in the TM,JCT and Schlemm tube of mice,eventually increase of outflowing resistance of aqueous humor.3.The increased IOP caused the activation of TGF-β/Smad signaling pathway,which may be the mechanism leading to the changes of ECM protein in the TM,JCT and Schlemm tube of mice.4.c-Ski gene inhibited TGF-β/Smad signaling pathway,and reconstructed ECM in the TM,JCT and Schlemm tube of mice,thus reduced outflowing resistance and lowered IOP. |
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