The Effect On Pathological Retinal Neovascularization By Regulating The Expression Of Telomerase Reverse Transcription

ObjectiveTo detect the expression of mTERT and to investigate the inhibitory effect of small interfering RNA(siRNA) targeting TERT on murine retinal neovascularization,and explore the feasibility of potential therapeutic approach in retinal vascular disease.Methods1.Expression of mouse telomerase reverse transcription in a mouse model of oxygen-induced retinopathy1.1.Establishment of oxygen-induced retinal neovascularization in mice.Sixty 7-day-old C57BL/6J mice were divided into oxygen-induced retinopathy group and control group without restriction of gender.In oxygen-induced retinopathy group,30 mice were exposed to 75±2%oxygen for 5 days and then to room air;In control group, 30 mice were raised in room air.1.2.Observation of the retinal neovascularization.The mice's vena caudalis were perfused with 2%Evens blue solution at P12,P14,P19,P21,P24,P30.Eyeballs were enucleated and fixed in 4%paraformaldehyde for half an hour.Then the retina was separated and flat-mounted on the slide.The morphologic changes of retinal vessel were observed and captured under fluorescence microscope.1.3.Reverse-transcription polymerase chain reaction(RT-PCR).In the each group,the retina were all carefully dissected on the postnatal day 19.The total RNA was isolated and cDNA was synthesized before RT-PCR was performed.The PCR products were separated by 2%agarose gel electrophoresis and photographed.1.4.Inhibition of TERT mRNA were tested by Real-time PCRThe same procedures of total RNA extraction and first-strand cDNA synthesis were performed as mentioned above.Each sample was assayed with RealMasterMix(TIANGEN BIOTECH(BEIJING) CO.,LTD,China).Fluorescent real-time quantitative polymerase chain reaction system(total 20μl) was made.The Fluorescent signals were detected at 60℃.The cycling conditions were as follows:5min at 95℃and 40 cycles of amplification for 15 s at 95℃and 1 min at 60℃.The quantification data were analyzed with the SDS System Software(7500,ABI,USA ).1.5.The expression of mTERT were confirmed by immunohistochemistry. At P19,4μm cross sections were made in the hyperoxia-exposed and normal retinas.Sections were incubated with rabbit anti-Human/Mouse/Rat Telomerase 60 minutes at 37℃.Anti-rabbit immunoglobulin G,depending on the primary antibody,was used as a secondary antibody for 30 min..Peroxidase activity was detected with the substrate diaminobenzidine.Permanent slides were covered with a 1.5-mm thick cover slip, examined using a light microscope and photographed.1.6.Histological observation and vascular endothelial cells counting.The eyeballs were enucleated killed and then fixed.After paraffin imbedding,4 urn serial slices,hematoxylin-eosin staining,select one section every 60μm to count the endothelial cell nucleus that break through the inner limiting membrane.2.TERT-siRNA inhibits oxygen-induced retinal neovascularization in mice2.1 Design and synthesis of TERT siRNATwo recombinant plasmids TERT siRNA(pSIREN-mTERT-1) and negative plasmid (pSIREN-mTERT-N) were constructed2.2 Inhibition of TERT gene in oxygen-induced retinopathy mice by injected intravitrously with TERTsiRNA 60 seven-day-old C57BL/6J mice were divided randomly into therapeutic group(A), negative plasmid group(B),oxygen-induced retinopathy group(c),20 mice in each group.Group A,B and C were exposed to 75%±2%oxygen for 5 days and then to room air,which induced mice retinal neovascularization.Group A and B were injected two kinds of the above recombinant plasmid into the murine vitreous on the 12th day.Mice of group C weren't injected intravitrously.2.3 Observation of the retinal neovascularization by angiographyOn the postnatal day 19,two samples of above 3 groups were taken.The mice's vena caudalis were perfused with 2%Evens blue solution.Eyeballs were enucleated and fixed in 4%paraformaldehyde for half an hour.Then the retina was separated and flat-mounted on the slide.The morphologic changes of retinal vessel were observed and captured under fluorescence microscope(Nicon Eclipase E800,Japan).2.4 Inhibition of TERT mRNA were tested by reverse-transcription polymerase chain reaction(RT-PCR)The retinas were all carefully dissected on the postnatal day 19 in group A,B,C.The same procedure of RT-PCR and real-time PCR were performed as mentioned above.Expression of TERT mRNA were confirmed by reverse-transcription polymerase chain reaction(RT-PCR)2.5 Inhibition of TERT mRNA were tested by Real-time PCRThe same procedures of total RNA extraction and first-strand cDNA synthesis were performed as mentioned above.Each sample was assayed with RealMasterMix.. Fluorescent real-time quantitative polymerase chain reaction system(total 20μl) was made.The Fluorescent signals were detected at 60℃.The quantification data were analyzed with the SDS System Software(7500,ABI,USA).2.6.Quantification of neovascular proliferative retinopathy examine the effects of siRNA on the retinal neovascularizationParaffin tissue slice with hematoxylin-eosin staining showed that the average counts of vascular endothelial cells which break through the inner limiting membrane in the above 3 group.The same procedure as above vascular endothelial cells counting.Histological observation and vascular endothelial cells counting were used to examine the effects of siRNA on the retinal neovascularization.2.7 Inhibition of telomerase detected by Western blot detectionThe retinas of group A,B,C were all carefully dissected on the postnatal day 19.After proteins of each group were extraction,total protein 25 uL was transferred into wells.The protein from gel was transfered to the NC membrane by an electrical transfer after electrophoresis.Then washed with TBST,the membrane was incubated in washing buffer containing 5%defatted milk for 1.5 h at 4℃.The membrane was incubated in 1000uL primary antibody(Rabbit Anti-Human/Mouse/Rat Telomerase polyclonal antibodyl:500 dilution)(ab23699,Abeam plc.332 Cambridge Science Park,Cambridge,CB4 OFW,UK) at room temperature for 1.5 h;HRP labeled goat anti—rabbit polyclonal antibody(1:3000 dilution)(KIT-9901-B,Maixin_Bio,China) was added at room temperature for 1h.Then wash with TBST solution,the membrane was treated with ECL chemical luminescent kit and developed in dark room.Blots were developed using a chemiluminescent solution,quantitatively assayed with Imagine J.Results1.Expression of mouse telomerase reverse transcription in a mouse model of oxygen-induced retinopathyThe nonperfused retina was central and the perfused retina was peripheral on P12.Most of the central retina showed almost no perfusion and the radial vessels appeared tortuous and dilated on P14.Retinal neovascularization occurred at maximum between postnatal day 17 and postnatal day 19.Retinal neovascularization began to recede from the postnatal day 21. Paraffin tissue slice with hematoxylin-eosin staining showed that in the control group the average Counts of vascular endothelial cells which break through the inner limiting membrane were hardly seen,but in hyperxia group were noticeably more than in the control group.Reverse-transcription polymerase chain reaction(RT-PCR) results:The mRNA of mTERT,bFGF and Bc1-2 in the retinopathy group were higher than in the control group,respectively(p<0.05).Real-time PCR results:The expression of mTERT mRNA in the retinopathy group was noticeably highcr than in the control group (p<0.05).Immunohistochemical staining showed that mTERT protein were positive in the retinal neovascularization of the hyperxia group,but were negtive in the retinal vessel of the control group.2.TERT-siRNA inhibits oxygen-induced retinal neovascularization in miceRetinal flat after Evans blue angiography indicated that the vessels of group A formed a fined radial branching pattern,which similar to normal mice.In group A,trie retinal neovascularization reduced and the structure of retina were more regular than group B and C.At the same time the large vessels were distorted,neovascular clusters proliferated and fluorescence leaked in the middle and periphery area in group B and C.RT-PCR and Real-time PCR showed the expression of TERT mRNA was downregulated in group A compared with groups B and C(P<0.05).The mRNA of bFGF was also downregulated in group A compared with groups B and C(P<0.05).Paraffin tissue slice with hematoxylin-eosin staining showed that the average counts of vascular endothelial cells which break through the inner limiting membrane in group A were less than group Band C,the differences were significant(P<0.05).Weastern blot showed the proteins of TERT were reduced after knocking down TERT gene in a mice model of oxygen-induced retinopathy,there were significant difference with group B and C(P<0.05).Conclusions:1.TERT may play an essential role in the development of pathological retinopathy which may be potential therapeutic target.2.Telomerase-dependent pathway may be took part in oxygen-induced neovascularization, and it may be related to anti-apoptosis Bel-2 gene.3.During the development of Pathology angiogenesis,TERT gene may be the downstream gene of bFGF.4.Retinal neovascularization was inhibited significantly by specific TERT siRNA,which may have great potential for use in clinical therapy.