Protective Effects And Mechanism Of C-Ski On Optic Never Injury In Glaucoma

Part oneEstablishment of N-methyl-D-aspartate?NMDA?-induced normal-tension glaucoma rat modelObjective:To explore the optimal scheme of N-methyl-D-aspartate?NMDA?-induced normal-tension glaucoma rat model.Methods:Thirty-six Sprague-Dawley male rats were randomly distributed into 4groups.Nine rats were in normal group?group A?.The rest of 27 rats were equally divided into 3 groups?groups B,C,D?to receive intravitreal injection of 3 different concentrations?5?l?of NMDA?20 nmol,40 nmol,and 80 nmol?into the right eyes.The left eyes received intravitreal injection of 5?l PBS used as a control?group E?.Three days,seven days and fourty days after the intravitreal injection of NMDA,all rats were killed and the eyes were enucleated.The ganglion cell complex?GCC?thickness was measured by Hematoxylin/eosin staining.The apoptosis index?AI?of cells in the ganglion cell layer?GCL?was detected by TUNEL staining.Results:1.Intravitreal injection of NMDA?groups B,C,D?caused GCC become thin around the optic disc in the rat retina,compared with un-injected rats in group A and E?P<0.05?,and group C is thinner than group B?P<0.05?.The thickness of GCC in group B,C,D on 14d is thinner than those on 3d and 7d?P<0.05?.There was no significant difference between group A and group E,between group B,C,D on 3d and 7d,and between group C and group D?P>0.05?.2.It is obvious that rats in groups B,C and D had a dramatically higher of cells AI in GCL,compared with those in group A and group E?P<0.05?,cells AI in GCL in group C is higher than that in group B?P<0.05?.The cells AI in GCL of group B,C,D on 14d is higher than those on 3d and 7d?P<0.05?.There was no obvious difference between group A and group E,group B,C,D on 3d and 7d,as well as group C and group D?P>0.05?.Conclusion:The optimal dosage of NMDA was 40nmol to establish normal-tension glaucoma rat model,and the best time of testing was 14 days after intravitreal injection of NMDA.Part twoConstruction of recombinant type II adeno-associated viruses-mediated c-Ski overexpression and interferenceObjective:To construct recombinant type II adeno-associated viruses?rAAV2?-mediated c-Ski overexpression and interference vectors and obtain the effective concentration of gene therapy.Methods:To pack recombinant type II adeno-associated viruses?rAAV2-c-Ski/GFP?,we overexpressed c-Ski and GFP gene at the same time and confirmed that the purpose protein expression in cell level.The recombinant type II adeno-associated viruses?rAAV2-GFP?only expressed GFP gene used as control.The virus titer was detected by Real-time PCR.We used the same methodtobuildrecombinanttypeIIadeno-associatedviruses?rAAV2?-mediated c-Ski interference of expression vectors.Results:The recombinant type II adeno-associated viruses?rAAV2?-mediated c-Ski overexpression vectors were constructed and confirmed by sequencing.Recombinant adeno-associated viruses rAAV2-c-Ski/GFP and rAAV2-GFP were produced in 239T cells and the titer of both viruses was 1.55×10¹²² v.g./ml.We packed type II adeno-associated viruses-mediated c-Ski interference virus?rAAV2-c-Ski-shRNA/GFP and rAAV2-NC-shRNA/GFP?with the same method,and the titers were 2.03×10¹³³ v.g./ml and 1.56×10¹³³ v.g./ml.Conclusion:We constructed recombinant type II adeno-associated viruses-mediated c-Ski overexpression and interference vectors successfully and confirmed the efficiency of virus titers in the 293T cells.Part three To study the effect of recombinant type II adeno-associated virus-mediated c-ski gene overexpression and interference on glaucomal optic nerve injury.Objective:To observe the transfection of recombinant type II adeno-associated virus?rAAV2?-mediated the c-Ski gene in the retina and explore the protective effects and possible mechanism of c-Ski on glaucomal optic nerve injury.Methods:Sixty Sprague-Dawley male rats were distributed into 5 groups:normal group?group A,n=12?;glaucoma group?group B,n=12?accepting intravitreal injection of 40 nmol NMDA;glaucoma+rAAV2-c-Ski/GFP group?group C,n=12?accepting intravitreal injection of NMDA plus intravitreal injection of rAAV2-c-Ski/GFP;glaucoma+rAAV2-GFP group?group D,n=12?accepting intravitreal injection of NMDA plus intravitreal injection of rAAV2-GFP and normal+rAAV2-c-Ski/GFP group?group E,n=12?accepting intravitreal injection of rAAV2-c-Ski/GFP.Another 60 rats were randomly distributed into 5 groups:normal group?group a,n=12?;glaucoma group?group b,n=12?accepting intravitreal injection of 40 nmol NMDA;glaucoma+rAAV2-c-Ski–shRNA/GFP group?group c,n=12?accepting intravitreal injection of NMDA plus intravitreal injection of rAAV2-c-Ski-shRNA/GFP;glaucoma+rAAV2-NC-sh RNA/GFP group?group d,n=12?accepting intravitreal injection of NMDA plus intravitreal injection of rAAV2-GFP and normal+rAAV2-c-Ski-sh RNA/GFP group?group e,n=12?accepting intravitreal injection of rAAV2-c-Ski-shRNA/GFP.Another 8 healthy Sprague-Dawley male rats were randomly divided into 2 groups:group G accepting intravitreal injection of rAAV2-GFP and group S accepting intravitreal injection of rAAV2-c-Ski/GFP.All rats in group C,E,S received intravitreal injection of 5?l rAAV2-c-Ski/GFP of 1.55×10¹²² v.g./ml in titer in right eyes.All rats in group c and group e received intravitreal injection of 5?l rAAV2-c-Ski-shRNA/GFP of 2.03×10¹³³ v.g./ml in titer in right eyes.All rats in group D and G received intravitreal injection of 5?l rAAV2-GFP of 1.55×10¹²v.g./ml in titer in right eyes.All rats in group d received intravitreal injection of5?l rAAV2-NC-shRNA/GFP of 1.56×10¹³³ v.g./ml in titer in right eyes.After two weeks,all rats in group B?b?,C?c?and D?d?received intravitreal injection of 5?l 40 nmol NMDA in right eye.At 4 weeks,Flash-visual evoked potential?F-VEP?and electroretinogram?ERG?were recorded under scotopic conditions to assess the retinal function.We observed the transfection of recombinant type II adeno-associated virus?rAAV2?–mediated the c-Ski gene in the retina by frozen section of the retina.Hematoxylin/eosin staining was performed on retina cross-sections for measurement of ganglion cell complex?GCC?thickness.Apoptosis index?AI?of cells in the ganglion cell layer?GCL?was assessed by TUNEL on retina cross-sections.The protein level of c-Ski,TGF-?2,Bax,Bcl-2,Smad2/3,Smad4,p-Smad2/3,FN,LN were detected by Western blotting.Results:1.Fluorescent microscopy found that rAAV2 mediated c-Ski genes transfected successfully in the retina?in groups G and S?.Most GFP+-transfected cells were located in the GCL.2.Compared with rats in group A?a?and group E?e?,there was a significant reduction in the amplitudes of the F-VEP amplitudes and b-waves of the ERG in the rats of intravitreal injection of NMDA?P<0.05?).Administration of rAAV2-c-Ski/GFP mitigated the reduction of the F-VEP amplitude and the b-waves amplitude of ERG in the glaucomatous eyes?P<0.05?.However,administration of rAAV2-c-Ski-shRNA/GFP aggravated the reduction of the b-waves amplitude of ERG and the F-VEP amplitude in the glaucomatous eyes?P<0.05?.3.Intravitreal injection of NMDA caused GCC become thinn in the rat retina,compared with normal rats in group A?a?and group E?e??P<0.05?.Administration of rAAV2-c-Ski/GFP mitigated the changes and significantly increased the thickness of GCC?P<0.05?,compared with rats in group B and D.But,administration of rAAV2-c-Ski-shRNA/GFP aggravated the changes and significantly thinned the thickness of GCC?P<0.05?,compared with rats in group b and d.4.It was evident that rats of group B?b?,group C?c?and group D?d?had a distinctly higher of TUNEL cells number of RGC,compared with rats in group A?a?and group E?e??P<0.05?.Administration of rAAV2-c-Ski/GFP mitigated the changes and significantly reduced the TUNEL cells in RGCs?P<0.05?,compared with ratsingroupBandgroupD.However,administrationof rAAV2-c-Ski-shRNA/GFP aggravated the changes and significantly increased the TUNEL cells in RGCs?P<0.05?,compared with rats in group b and d.5.The protein level of c-Ski,TGF-?2,Bax,Bcl-2,Smad2/3,Smad4,p-Smad2/3,FN,LN was detected by Western blotting.The expression of c-Ski was increased in the eyes that received intravitreal injection of rAAV2-c-Ski/GFP?P<0.05?.However,the expression of c-Ski was decreasedintheeyesthatreceivedintravitrealinjectionof rAAV2-c-Ski-shRNA/GFP?P<0.05?.There was a marked decrease in the level of Bcl-2 with a concomitant increase in the level of Bax protein in the eyes of group B?b?,group C?c?and group D?d?,relative to the rats in group A?a?and group E?e??P<0.05?.Moreover,the expression of TGF-?2,p-Smad2/3,FN,LN in group B?b?,group C?c?and group D?d?was significant increased,compared with rats in group A?a?and group E?e??P<0.05?.In group C,the eyes received intravitreal injection of rAAV2-c-Ski/GFP caused the protein level of TGF-?2,p-Smad2/3,FN,LN reduced,relative to rats in group B and group D?P<0.05?.However,the eyes of group c received intravitreal injection of rAAV2-c-Skish-RNA/GFP caused the protein level of TGF-?2,p-Smad2/3,FN,LN further increased,compared with the rats in group b and group d?P<0.05?.Conclusion:1.The rAAV2 successfully mediated the expression of c-Ski gene in the retina,and the positive transfection cells were mainly located in the retinal of GCL.2.Intravitreal injection of rAAV2-c-Ski/GFP could reduce the RGC apoptosis induced by NMDA.It may be a candidate therapy for neuroprotective in the treatment of glaucoma.The possible mechanism is that c-Ski inhibits TGF-?/Smad signaling pathway via disturbing Smad2 and Smad3phosphorylation,causing the retinal ECM deposition reduction to control the RGC apoptosis.