摘要
目的
研究高氧诱导小鼠视网膜新生血管模型中端粒酶逆转录酶(telomerasereverse transcription,TERT)表达水平是否有变化,为进一步研究视网膜新生血管疾病的预防和治疗提供新的靶点。在此基础上,探讨小鼠端粒酶逆转录酶(TERT)小分子干扰RNA(siRNA)对小鼠视网膜新生血管形成的抑制作用,及其用于视网膜新生血管疾病治疗的可行性。
材料和方法
1.高氧诱导小鼠视网膜新生血管模型中端粒酶逆转录酶的表达变化
1.1动物模型的建立
健康C57BL/6J新生小鼠60只,性别不限,随机分为高氧模型组与正常对照组,每组30只。高氧模型组小鼠取生后7天C57BL/6J与其母鼠一起进入自制的密闭氧舱。氧箱内通入湿润氧气,每日检测氧浓度2次,维持氧箱内氧气的体积分数为(75±2)%。5天后将小鼠返回正常大气环境中饲养。正常对照组小鼠与其母鼠生后一直置于正常大气环境中饲养。所有小鼠均于自然光照明下饲养,昼夜正常交替,室温控制在23℃±2℃。
1.2视网膜新生血管的观察
于小鼠生后12、14、19、21、24、30天时,分别取高氧模型组和正常对照组各2只小鼠(4眼),麻醉后经尾静脉注射2%伊文思蓝(Evans blue)溶液,待大约5分钟后观察小鼠四肢末端及口鼻耳处变蓝后处死小鼠,摘取眼球,置4%多聚甲醛固定半小时后,小心分离视网膜组织并铺片,荧光显微镜下观察视网膜血管情况。
1.3.RT-PCR法检测mTERT及bFGF、Bc1-2 mRNA的表达
分别取生后19天高氧组及对照组小鼠各5只,麻醉后摘除眼球,小鼠颈椎脱臼处死,将摘除的眼球去除眼前节组织及玻璃体,分离出视网膜组织并提取视网膜总RNA,反转录成cDNA后,进行RT-PCR扩增。琼脂糖凝胶电泳并用凝胶电泳照相仪照相。用Image J分析软件测量目的基因电泳条带的灰度值,与内参GAPDH灰度值做比较,灰度比值=目的基因灰度值/GAPDH灰度值。
1.4 Real-time法检测mTERT mRNA的表达
提取P19小鼠视网膜总RNA,反转录成cDNA(同RT-PCR),用Primer 5软件设计引物,配制荧光定量RT-PCR反应体系(总计20μl),设荧光定量条件,在60℃检测荧光信号,分析图像。
1.5.免疫组化
于小鼠生后19天时,分别取高氧模型组和正常对照组各2只小鼠(4眼),腹腔内注射麻醉后处死小鼠,摘取眼球,置固定液固定3小时移至10%甲醛过夜,石蜡固定切片后,60℃烤片过夜,脱蜡,PBS洗。EDTA微波修复,3%H_2O_2水溶液微波1分钟,吸干水渍,加封闭血清,封闭10-15分钟。37℃水浴,加一抗RabbitAnti-Human/Mouse/Rat Telomerase(1:200),置37℃水浴60分钟,PBS洗,加增强剂15分钟,37℃水浴PBS洗,加酶标羊抗鼠/兔IgG聚合物二抗30分钟,PBS洗三次,DAB缓冲液染片。苏木素轻度复染,脱水,透明,中性胶封片,观察时置于显微镜下观察、照相。
1.6组织切片观察
处死高氧模型组和正常对照组生后19 d幼鼠各3只(6只眼),摘下眼球。去除晶状体,4%中性甲醛溶液过夜固定。梯度乙醇脱水二甲苯透明3～4分钟,65℃浸蜡2小时,石蜡包埋。全眼球连续矢状位切片,厚度4μm,每隔60μm(15张切片)取1张组织贴片于一张玻片。HE染色:常规石蜡切片,烤片箱60℃烤片过夜后,二甲苯脱蜡3,梯度乙醇复水,行HE染色。时间:苏木素染色15分钟,1%盐酸乙醇分化1～2分钟,自来水洗,1%氨水返蓝数秒,自来水30分钟,1%伊红染色3分钟,梯度乙醇脱水,二甲苯透明,中性树胶封片。光学显微镜下观察视网膜血管形态,观察突破内界膜的内皮细胞核数(含有视乳头的切片弃去)。
2.TERT基因siRNA抑制小鼠视网膜新生血管形成的实验研究
2.1 RNAi重组质粒构建、扩增、提取
根据基因库中mTERT基因(AF_051911)的mRNA序列设计siRNA作用靶点,化学合成(由宝生物工程(大连)有限公司设计构建)两种质粒。mTERT siRNA重组质粒pSIREN-mTERT-1和阴性对照质粒pSIREN-mTERT-N,siRNA序列分别为:5'-CAGATCAAGAGCAGTAGTC-3'和5'-TTAGCGACGATAAGGCAAC -3'。表达载体均为pSIREN-DNR-DsRed-Express。
2.2新生小鼠玻璃体腔注射
选择7d龄C57BL/6J小鼠60只随机数字法分为基因治疗组(A)、阴性质粒对照组(B)、高氧对照组(C)置于75%±2%高氧环境中生活5天,然后回到正常氧环境中。于第12天出氧舱时,分别向基因治疗组(A)、阴性质粒对照组(B)两组小鼠玻腔内注射上述两种质粒。高氧对照组(C)不予玻璃体腔注射。
2.3视网膜Evens blue荧光造影铺片观察
从各组中随机取出幼鼠P19 2只,麻醉后用2%Evens blue灌注进行视网膜铺片,摘取眼球置于4%多聚甲醛溶液固定。固定半小时后在显微镜下,沿角巩膜剪开眼球,去除角膜、晶状体,小心娩出视网膜,将视网膜以视乳头为中心,放射状切开4刀,置于干净载玻片上,铺平,滴中性胶一滴,盖上盖玻片,观察各组小鼠视网膜血管形态变化
2.4 RT-PCR法检测mTERT及bFGF mRNA的表达变化
分别取生后19天各组小鼠,麻醉后摘除眼球,小鼠颈椎脱臼处死,将摘除的眼球去除眼前节组织及玻璃体,分离出视网膜组织并提取视网膜总RNA,反转录成cDNA后,进行RT-PCR扩增。2%琼脂糖凝胶电泳,并用凝胶电泳照相仪照相。用ImageJ分析软件测量目的基因电泳条带的灰度值,与内参GAPDH灰度值做比较,灰度比值=目的基因灰度值/GAPDH灰度值。
2.5 Real-PCR法检测mTERT mRNA的表达变化
提取各组P19小鼠视网膜总RNA,反转录成cDNA(同RT-PCR),用Primer 5软件设计引物,配制荧光定量RT-PCR反应体系(总计20μl),设荧光定量条件,在60℃检测荧光信号,分析图像。
2.6组织切片观察及血管内皮细胞计数
处死各组P19幼鼠3只,摘下眼球。4%中性甲醛溶液过夜固定。梯度乙醇脱水,二甲苯透明,65℃浸蜡,石蜡包埋。做与视神经矢状轴平行的视网膜连续切片(厚度4μm),每隔60μm(15张切片)取一张,每只眼选取10张切片,含视神经的切片弃去。HE染色:烤片箱过夜烤片后,二甲苯脱蜡,梯度乙醇水化,行HE染色。时间:苏木素染色15分钟,1%盐酸乙醇分化1～2分钟,自来水洗,1%氨水返蓝数秒,自来水30分钟,1%伊红染色3分钟,梯度乙醇脱水,二甲苯透明,中性树胶封片。光学显微镜下观察视网膜血管形态,观察计数突破内界膜的内皮细胞核数,计算各组内皮细胞核平均数,进行定量分析比较。
2.7 Weastern blot检测mTERT蛋白的表达变化
处死各组P19幼鼠3只,摘下眼球。分离视网膜组织,利用研钵粉碎组织块,加入RIPA缓冲液60,使用前加入PMSF(100:1),使PMSF的最终浓度为1mM.玻璃棒匀浆,直至充分裂解。移入离心管4℃约20,000 g(约15,000转)5分钟。取相同质量的细胞裂解液(体积*蛋白质浓度),并加1/3体积的4×电泳加样缓冲液,沸水浴中5分钟,上样、电泳、转膜,漂洗,加一抗、二抗孵育,蛋白检测。
结果
1.高氧诱导小鼠视网膜新生血管模型中端粒酶逆转录酶的表达变化
高氧诱导模型小鼠P12后极部出现大片无灌注区,P14后极部出现新生血管迂曲、渗漏等视网膜血管病变。P17-19新生血管形成达到高峰,P21-30逐渐减少。正常P19小鼠视网膜组织切片HE染色基本看不到突出内界膜的血管芽及血管管腔,内界膜下视网膜内的血管内皮细胞核散在分布、数量较少;高氧组P19小鼠视网膜组织切片见大量突出内界膜伸向玻璃体腔的血管管腔及血管芽,内界膜下视网膜内也有大量血管内皮细胞增生。RT-PCR:19天高氧模型组小鼠视网膜mTERT及bFGFBc1-2 mRNA表达较同日龄正常对照组小鼠明显提高,二者有显著性差异(p<0.05)Real-time PCR:P19高氧模型组小鼠视网膜mTERT mRNA表达较同日龄正常对照组小鼠明显上调,有显著性差异(p<0.05)。免疫组织化学检测结果:P19高氧诱导小鼠视网膜新生血管模型中视网膜新生血管mTERT表达阳性,对照组同日龄新生小鼠视网膜血管mTERT表达阴性。
2.TERT基因siRNA抑制小鼠视网膜新生血管形成的实验研究
荧光造影视网膜铺片显示,A组整个视网膜血管分布网基本正常,走形较自然,基本接近正常对照组,未见明显的新生血管丛及大片荧光渗漏,只在视网膜中周部及周边部见少许荧光渗漏,但较B、C组明显减少。B、C组视网膜血管紊乱,中周部血管迂曲,伴大片荧光渗漏。RT-PCR及Real-time PCR显示A组小鼠视网膜TERTmRNA表达明显减少,与B、C组有统计学差异(P<0.05)。A组小鼠P19视网膜bFGFmRNA与B、C组没有统计学差异(P>0.05)。组织切片HE染色观察,A组仅见1处新生血管芽,偶见突出内界膜的细胞核;B、C组见散在突出内界膜伸向玻璃体腔的血管芽,内界膜下出现明显的血管内皮细胞增生;光镜下观察突破内界膜新生血管内皮细胞计数,A组较B、C组明显减少,有统计学差异(P<0.05)。Weastern Blot检测A组TERT蛋白较B、C组明显降低,均有统计学差异(P<0.05)。
结论
1.TERT对视网膜新生血管的形成起重要调控作用,可能会成为视网膜新生血管疾病预防和治疗的新靶点。
2.端粒酶-依赖途径可能参与了视网膜新生血管的形成,其促进视网膜新生血管形成的机制可能与抗凋亡因子Bc1-2的高表达有关。
3.在视网膜新生血管形成过程中,基因沉默TERT基因并不能下调bFGF基因的表达,TERT有可能是bFGF的下游基因。
4.TERT特异的siRNA能有效地抑视网膜新生血管动物模型视网膜中视网膜新生血管的形成,可能会成为一种治疗视网膜新生血管疾病的新方法。
Objective
To 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.
Methods
1.Expression of mouse telomerase reverse transcription in a mouse model of oxygen-induced retinopathy
1.1.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 PCR
The 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 mice
2.1 Design and synthesis of TERT siRNA
Two recombinant plasmids TERT siRNA(pSIREN-mTERT-1) and negative plasmid (pSIREN-mTERT-N) were constructed
2.2 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 angiography
On 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 PCR
The 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 neovascularization
Paraffin 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 detection
The 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.
Results
1.Expression of mouse telomerase reverse transcription in a mouse model of oxygen-induced retinopathy
The 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 mice
Retinal 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.
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