Twist基因干扰抑制猴视网膜微血管内皮细胞的迁移

目的 观察Twist基因干扰对体外培养的猴视网膜微血管内皮细胞(RF/6A)迁移及磷酸化Akt(pAkt)蛋白表达的影响.方法 体外培养的RF/6A细胞系,并构建Twist干扰质粒和对照质粒.分为Twist干扰质粒组、阴性对照组、磷酸盐缓冲液(PBS)组,各组应用脂质体基因转染方法,转入相应的质粒表达载体,采用Transwell小室法检测发生迁移的内皮细胞并计数;铺Matrigel胶,行内皮细胞体外成管实验,观察Twist基因干扰对内皮细胞成管的抑制作用;蛋白质免疫印迹检测Twist基因干扰对RF/6A细胞pAkt蛋白表达的影响.结果 Transwell小室计数结果表明,转染Twist干扰质粒后,迁移的细胞数显著低于阴性对照组和PBS组(F=23.786,P=0.000).体外成管实验结果表明,Twist干扰质粒组内皮细胞成管数显著低于阴性对照组和PBS组(F=7.159,P=0.014).Ttwist干扰质粒组的pAkt蛋白表达明显减少.结论 Twist基因干扰可显著抑制视网膜微血管内皮细胞的迁移,可能机制是通过抑制pAkt蛋白的表达发挥作用.     

Twist基因干扰抑制猴视网膜微血管内皮细胞的迁移

目的 观察Twist基因干扰对体外培养的猴视网膜微血管内皮细胞(RF/6A)迁移及磷酸化Akt(pAkt)蛋白表达的影响.方法 体外培养的RF/6A细胞系,并构建Twist干扰质粒和对照质粒.分为Twist干扰质粒组、阴性对照组、磷酸盐缓冲液(PBS)组,各组应用脂质体基因转染方法,转入相应的质粒表达载体,采用Transwell小室法检测发生迁移的内皮细胞并计数;铺Matrigel胶,行内皮细胞体外成管实验,观察Twist基因干扰对内皮细胞成管的抑制作用;蛋白质免疫印迹检测Twist基因干扰对RF/6A细胞pAkt蛋白表达的影响.结果 Transwell小室计数结果表明,转染Twist干扰质粒后,迁移的细胞数显著低于阴性对照组和PBS组(F=23.786,P=0.000).体外成管实验结果表明,Twist干扰质粒组内皮细胞成管数显著低于阴性对照组和PBS组(F=7.159,P=0.014).Ttwist干扰质粒组的pAkt蛋白表达明显减少.结论 Twist基因干扰可显著抑制视网膜微血管内皮细胞的迁移,可能机制是通过抑制pAkt蛋白的表达发挥作用.     

重组腺病毒-p21抑制猴视网膜微血管内皮细胞增生的作用研究

目的 观察重组腺病毒-p21 (rAd-p21)对体外培养的猴视网膜微血管内皮细胞(RF/6A)增生的作用。方法 体外培养RF/6A细胞系,分为磷酸盐缓冲液(PBS)组、rAd-p21转染组及阴性对照组,并转入相应的质粒表达载体。应用逆转录聚合酶链反应(RT-PCR)法及蛋白免疫印迹(Western blot)检测p21mRNA及蛋白在RF/6A细胞中的表达;应用流式细胞仪检测p21基因对RF/6A细胞周期的影响;行内皮细胞体外成管实验观察p21基因对RF/6A细胞成管的抑制作用。结果 rAd-p21转染组p21 mRNA及蛋白表达显著增高。细胞周期检测结果显示,PBS组、rAd-p21转染组、阴性对照组G0/G1期细胞百分比分别为（40.76±6.66）％、（67.45±11.61）％、(41.55±8.99)％;rAd-p21转染组RF/6A细胞出现G0/G1期阻滞,G0/G1期细胞比例增多。rAd-p21转染组与PBS组和阴性对照组比较,差异有统计学意义(F=21.284,P=0.000)。体外成管实验显示,PBS组、rAd-p21转染组、阴性对照组每一视野下内皮细胞成管数分别为（8.25±3.19）、（3.86±1.21）、(7.62±2.69)个;rAd-p21转染组与PBS组和阴性对照组比较,差异有统计学意义(F=7.138,P=0.004)。结论 rAd-p21可成功转染RF/6A细胞并稳定表达p21 mRNA及蛋白,并可显著抑制其增生。     

血管能抑素基因转移抑制视网膜新生血管的实验研究

目的 观察血管能抑素真核表达质粒(pCMV-HA)对小鼠视网膜新生血管RNV形成的抑制作用.方法 将鼠龄为7 d的56只C57BL/6J新生小鼠随机分为正常对照组、氧诱导视网膜病变(OIR)模型组、治疗组和空载体组,每组14只.后3组小鼠置于(75±2)%浓度的氧环境中饲养5 d后,回到正常空气环境中建立氧诱导的RNV动物模型.治疗组小鼠在出生后第12天出氧箱时行玻璃体腔注射血管能抑素pCMV-HA,空载体组注射等量空质粒.出生后第17天行伊凡思蓝(Evans blue)灌注血管造影视网膜铺片观察血管变化.石蜡切片行苏木精-伊红染色,光学显微镜下观察并计数突破视网膜内界膜的血管内皮细胞核数.结果 视网膜铺片结果显示,治疗组较OIR模型组和空载体组视网膜血管分布均匀,新生血管和无灌注区显著减少.治疗组突破内界膜的内皮细胞核数与OIR模型组及空载体组比较,差异有统计学意义(F=39.006,P<0.001).结论 血管能抑素pCMV-HA对氧诱导的RNV有显著的抑制作用.     

血管能抑素基因转移抑制视网膜新生血管的实验研究

Objective To observe the inhibitory effects of gene transfer of canstatin on retinal neovascularization in mice. Methods Fifty-six 7-day-old C57BL/6J mice were randomly divided into control group, oxygen-induced retinopathy (OIR) group, empty vector group and treated group, 14 mices in each group. Except for the control group, the mice in the other groups were exposed to (75±2)% oxygen for 5 days and then back to the normal air to establish the model of OIR. On postnatal 12 day, the treated group was received intravitreal injection of canstatin pCMV-HA, while the empty vector group was received the same volume of empty plasmid. The changes of retinal vessels were observed by Evans blue angiography on postnatal 17 day. With parafin section which stained by hematoxylin and eosin, then the number of endotheliocyte nuclei breaking throuhgh the internal limiting membrane(ILM) was observed and counted by optical microscope. Results Retinal blood vessels distributed regularly in treated group compared with OIR group and empty vector group. The differences of the number of endotheliocyte nuclei breaking throuhgh ILM in treated group was significant compared with the other two groups(F= 39. 006, P< 0. 001).Conclusion The canstatin pCMV-HA can effectively inhibit the retinal neovascularization in OIR.     

血管能抑素基因转移抑制视网膜新生血管的实验研究

Objective To observe the inhibitory effects of gene transfer of canstatin on retinal neovascularization in mice. Methods Fifty-six 7-day-old C57BL/6J mice were randomly divided into control group, oxygen-induced retinopathy (OIR) group, empty vector group and treated group, 14 mices in each group. Except for the control group, the mice in the other groups were exposed to (75±2)% oxygen for 5 days and then back to the normal air to establish the model of OIR. On postnatal 12 day, the treated group was received intravitreal injection of canstatin pCMV-HA, while the empty vector group was received the same volume of empty plasmid. The changes of retinal vessels were observed by Evans blue angiography on postnatal 17 day. With parafin section which stained by hematoxylin and eosin, then the number of endotheliocyte nuclei breaking throuhgh the internal limiting membrane(ILM) was observed and counted by optical microscope. Results Retinal blood vessels distributed regularly in treated group compared with OIR group and empty vector group. The differences of the number of endotheliocyte nuclei breaking throuhgh ILM in treated group was significant compared with the other two groups(F= 39. 006, P< 0. 001).Conclusion The canstatin pCMV-HA can effectively inhibit the retinal neovascularization in OIR.     

Twist基因干扰抑制小鼠视网膜新生血管形成的研究

目的 通过RNA干扰技术抑制小鼠视网膜新生血管形成过程中Twist基因的表达,观察视网膜新生血管内皮细胞的迁移变化和细胞转型规律,寻找抑制视网膜新生血管发生的新靶点.方法 实验研究.取健康C57BL/6J新生小鼠建立高氧诱导小鼠视网膜新生血管动物模型.小鼠生后第12天,分别给予Twist干扰质粒和无关对照质粒玻腔注射治疗.第17天取材,行视网膜Evans蓝灌注铺片、组织病理学检查、新生血管内皮细胞计数和免疫组织化学检测及Real-Time PCR检测.对各组视网膜新生血管内皮细胞计数和Real-Time PCR检测目标基因的表达变化,采用单因素方差分析进行统计学比较.结果 光镜下观察突破内界膜的视网膜新生血管,正常对照组、高氧诱导组、干扰质粒组和对照质粒组突破内界膜的血管内皮细胞平均每眼分别为0.34±0.11、32.73±6.38、4.56±2.02和20.17±6.49.小鼠视网膜Evans蓝灌注铺片观察和石蜡切片HE染色观察显示Twist干扰质粒组小鼠第17天较高氧诱导组视网膜血管迂曲渗漏减轻,新生血管明显减少.Twist干扰质粒组视网膜新生血管内皮细胞计数较高氧诱导组显著减少.免疫组织化学检测可见Twist干扰质粒组小鼠视网膜Twist和波形蛋白表达较高氧诱导组明显减少.使用Real-Time PCR方法检测各组小鼠第17天视网膜Twist基因和波形蛋白基因的表达,Twist干扰质粒组表达较高氧诱导组下调(F=27.214,31.211;P＜0.05).结论 在小鼠视网膜新生血管形成过程中,细胞转型调控的Twist基因发挥重要作用,通过RNA干扰技术抑制Twist基因的表达,可阻遏细胞转型的发生,抑制视网膜新生血管的形成.

Abstract：

Objective The purpose of this research is to find the law of neovascular endothelial cell migration and transition through repressing the expression of Twist in mouse's retinal neovascularization with RNAi, and get a new target of inhibit retinal neovascularization. Methods Oxygen-induced retinopathy (OIR) was produced in new bom C57BL/6J mice by exposing postnatal day 7 (P7) pups to 75% oxygen for 5 days. P12 pups were injected 1 μl pTwist. siRNA plasmid solution or 1 μl negtive siRNA plasmid solution into vitreous cavity. Eyeballs were enucleated for Evans blue angiography, histopathologic examination,neovascular endothelial cell counting, immunohistochemistry and Real-Time PCR. Results Observed by light microscopy retinal neovascularization, the number of vascular endothelial cells per eye were 0.34±0.11,32.73±6.38, 4.56±2.02 and 20.17±6.49 in the normal control group, hyperoxia group,Twist plasmid group and the control plasmid group. Mouse retinal Evans blue perfusion and HE staining of paraffin sections showed that retinal vascular leakage, tortuous and angiogenesis significantly reduced in Twist plasmid group compared with hyperoxia group. Endothelial cell count was significantly decrease in Twist plasmid group. Both immunohistochemistry and real time PCR proved that Twist and vimentin expression in hyperoxia group were significantly higher than that of Twist plasmid group ( F=27.214,31.211 ;P＜0.05). Conclusion As mice retinal neovasculars growth, Twist may play important roles as a cell transition regulatory factor. Repressing Twist with RNAi, we can repress cell transition and inhibit retinal neovascular.     

鼠视网膜新生血管发生过程中Twist基因的表达

目的 探讨视网膜新生血管发生过程中Twist基因的表达情况.方法 对照实验研究.将40只新生C57/BL小鼠分为两组:(1)对照组,正常空气环境中饲养;(2)高氧组,在高氧环境中制作小鼠缺氧性视网膜新生血管动物模型.将出生第7天的小鼠放人氧箱,在75%浓度的高氧环境中饲养5 d后,取出氧箱.待出氧箱第12天和第17天(视网膜新生血管发生高峰时间)分别处死小鼠,制作视网膜铺片和切片,观察小鼠视网膜新生血管发生情况.应用免疫组织化学染色检测小鼠视网膜血管内皮细胞(VE)-钙黏蛋白、波形蛋白及Twist基因表达变化情况;提取全视网膜RNA,应用逆转录聚合酶链反应(RT-PCR)法检测VE-钙黏蛋白、波形蛋白及Twist基因表达变化情况.应用SPSS 11.5统计学软件进行数据处理,对高氧组与对照组小鼠出生后不同时间的VE-钙黏蛋白、波形蛋白、Twist基因表达的灰度值比较,采用两因素析因设计的方差分析,以P<0.05作为差异有统计学意义.结果 免疫组织化学检测结果显示,出生后第17天的小鼠,高氧组VE-钙黏蛋白表达的灰度值(65.19±8.39)与对照组(75.36±7.04)相比明显减少(F=8.616,P=0.009),波形蛋白表达的灰度值(95.09±14.13)与对照组(82.14±6.32)相比明显升高(F=6.999,P=0.016),Twist基因表达的灰度值(119.48±7.90)与对照组(93.30±6.37)相比亦明显升高(F=66.557,P=0.000).RT-PCR检测结果显示,不同处理组间波形蛋白、Twist基因表达的灰度值与检查时间存在交互作用(F=5.508,P=0.032;F=17.760,P=0.001).结论 在小鼠视网膜新生血管形成过程中,细胞转型调控的Twist基因发挥着重要作用,其作用机制可能是通过下调血管内皮细胞间的紧密连接蛋白,促进内皮细胞转型实现.     

重组腺病毒介导p21对小鼠视网膜新生血管生成的抑制作用

目的 观察重组腺病毒-p21 (rAd-p21)对氧诱导小鼠视网膜新生血管(RNV)的抑制作用.方法 将56只健康7日龄C57BL/6J小鼠随机分为对照组、磷酸盐缓冲液(PBS)组、rAd-p21组及rAd-无目的基因对照(rAd-NC)组,每组14只.PBS组、rAd-p21组及rAd-NC组建立氧诱导RNV模型,并于小鼠11日龄时玻璃体腔分别注入1μl PBS、rAd-p21及rAd-NC.对照组不做任何处理.小鼠17日龄时,每组处死4只小鼠,摘取眼球分别作荧光视网膜铺片和切片,观察小鼠RNV发生情况;Image-Pro plus 6.0软件测量分析无灌注区面积;同时提取视网膜总RNA和总蛋白,应用逆转录聚合酶链反应(RT-PCR)及蛋白免疫印迹法(Western blot)检测p21、细胞周期素依赖蛋白激酶2(CDK2) mRNA及蛋白在视网膜组织中的表达.结果 荧光及光学显微镜观察发现,rAd-p21组小鼠视网膜无灌注区、新生血管及突破视网膜内界膜的血管内皮细胞核较PBS组和rAd-NC组减少;rAd-p21组无灌注区面积较PBS组和rAd-NC组明显减少,组间差异有统计学意义(F=101.634,P＜0.05).RT-PCR及Western blot检测结果显示,rAd-p21组p21 mRNA和蛋白表达明显高于对照组、PBS组及rAd-NC组,组间差异有统计学意义(F＝839.664、509.817,P＜0.05);rAd-p21组CDK2 mRNA和蛋白表达明显低于对照组、PBS组及rAd-NC组,组间差异也有统计学意义(F=301.858、592.882,P＜0.05).结论 rAd-p21可通过上调p21表达、降低CDK2表达,抑制RNV生成.     

15-脂氧合酶-1基因转移抑制小鼠视网膜新生血管的实验研究

目的 观察15-脂氧合酶-1(15-LOX-1)基因转移对氧诱导小鼠视网膜新生血管的抑制作用.方法 7日龄C57BL/6J小鼠96只,随机分为正常对照组、氧诱导视网膜病变(OIR)模型组、基因治疗组和空白载体组.将小鼠与哺乳母鼠共同置于氧浓度为(75±2)％的氧箱内饲养5d后转移至正常环境中饲养5d,建立OIR模型.小鼠出生后第12天基因治疗组玻璃体腔注射携带增强型绿色荧光蛋白(EGFP)和小鼠15-LOX-1基因的重组腺病毒(Ad-15-LOX-1-EGFP)载体1μl;空白载体组注射等量携带EGFP的重组腺病毒(Ad-EGFP)载体.注射后第2天行视网膜铺片荧光显微镜观察EGFP的表达.注射后第5天行免疫荧光染色法、实时荧光定量聚合酶链反应和蛋白免疫印迹法检测15-LOX-1基因转染视网膜的表达;视网膜铺片观察视网膜血管变化,测量视网膜无灌注区和新生血管的相对面积;石蜡切片苏木精-伊红染色并计数突破视网膜内界膜的血管内皮细胞核.结果 Ad-15-LOX-1-EGFP注射第2天,视网膜铺片上观察到EGFP的表达.免疫荧光染色结果显示,15-LOX-1基因转染视网膜主要表达在外丛状层、内核层和神经节细胞层.基因治疗组15-LOX-1蛋白和mRNA表达水平明显高于OIR模型组和空白载体组,差异有统计学意义(t蛋白表达水平=22.74、24.13,tmRNA表达水平=12.51、13.40;P＜0.01);基因治疗组视网膜无灌注区和新生血管面积较OIR模型组和空白载体组显著减小,差异有统计学意义(t血管区面积=16.22、14.31,t新生血管面积=9.97、9.07;P＜0.01);基因治疗组中突破视网膜内界膜的血管内皮细胞核与OIR模型组和空白载体组比较明显减少,差异有统计学意义(t=14.25、11.62,P＜0.01).结论 15-LOX-1基因转移不仅可以减少氧诱导小鼠视网膜无灌注区面积,并且对视网膜新生血管有显著的抑制作用.     

重组腺相关病毒-色素上皮衍生因子抑制氧诱导小鼠视网膜新生血管的作用

Objective To investigate the effects of recombinant adeno-associated virus type-2 (rAAV2) mediated delivery of pigment epithelium-derived factor (PEDF) on oxygen-induced retinal neovascularization (OIRNV) in mice. Methods A total of 22 C57/BL6 mice at the age of 3 days received intravitreal injections of 1 μl rAAV2-PEDF and rAAV2-EGFP into the left eyes (experimental group) and the right eyes (control group). All mice were put into the oxygen box right after the injection to induce the OIRNV model. 4 mice were sacrificed and PEDF protein in retina was measured by western blot at postnatal days 13 (P13). Twelve mice underwent retinal angiography with high molecular weight fluorescein-dextran,and another 6 mice were sacrificed for retinal lectin immunohistochemistry staining at P17. Absolute and relative non-perfusion areas of retinal neovascularization were analyzed by Image-Pro Plus 5.1 software.Results The expression level of PEDF protein was higher in the experimental group than that in the control group. The absolute non-perfusion area was (0. 96 + 0.22) mm2 in the experimental group and (1.96±0. 34) mm2 in the control group; the difference between the two groups was significant (t = -8. 554, P<0.01). The relative non-perfusion area was (8. 64 ± 1.52) % in the experimental group and (17. 27 ± 2. 98)% in the control group with a significant difference between the two groups (t = -8. 97, P<0. 01). The absolute area of retinal neovascularization was (0. 37 ± 0. 11) mm2 in the experimental group which was obviously higher than (1.26±0. 38) mm2 in the control group (t=-7. 8, P<0. 01); the relative areas in experimental and control groups was (3. 96 ± 0. 66) % and ( 11.45 ± 2. 06) %, respectively, whose difference is apparently (t=-8. 51, P<0. 01). The areas of retina neovascularization were (0. 11±0. 003)mm2 and (0.41±0.02)mm2 in the experimental and control groups, respectively, and the differencebetween the two groups was significant (t =- 5.14, P< 0. 01). Conclusions PEDF protein can stably express in the mice retina after rAAV2-PEDF transfetion, rAAV2-PEDF can decrease the retinal nonperfusion areas and inhibit the retinal neovascularization in OIRNV mice.     

重组腺相关病毒-色素上皮衍生因子抑制氧诱导小鼠视网膜新生血管的作用

Objective To investigate the effects of recombinant adeno-associated virus type-2 (rAAV2) mediated delivery of pigment epithelium-derived factor (PEDF) on oxygen-induced retinal neovascularization (OIRNV) in mice. Methods A total of 22 C57/BL6 mice at the age of 3 days received intravitreal injections of 1 μl rAAV2-PEDF and rAAV2-EGFP into the left eyes (experimental group) and the right eyes (control group). All mice were put into the oxygen box right after the injection to induce the OIRNV model. 4 mice were sacrificed and PEDF protein in retina was measured by western blot at postnatal days 13 (P13). Twelve mice underwent retinal angiography with high molecular weight fluorescein-dextran,and another 6 mice were sacrificed for retinal lectin immunohistochemistry staining at P17. Absolute and relative non-perfusion areas of retinal neovascularization were analyzed by Image-Pro Plus 5.1 software.Results The expression level of PEDF protein was higher in the experimental group than that in the control group. The absolute non-perfusion area was (0. 96 + 0.22) mm2 in the experimental group and (1.96±0. 34) mm2 in the control group; the difference between the two groups was significant (t = -8. 554, P<0.01). The relative non-perfusion area was (8. 64 ± 1.52) % in the experimental group and (17. 27 ± 2. 98)% in the control group with a significant difference between the two groups (t = -8. 97, P<0. 01). The absolute area of retinal neovascularization was (0. 37 ± 0. 11) mm2 in the experimental group which was obviously higher than (1.26±0. 38) mm2 in the control group (t=-7. 8, P<0. 01); the relative areas in experimental and control groups was (3. 96 ± 0. 66) % and ( 11.45 ± 2. 06) %, respectively, whose difference is apparently (t=-8. 51, P<0. 01). The areas of retina neovascularization were (0. 11±0. 003)mm2 and (0.41±0.02)mm2 in the experimental and control groups, respectively, and the differencebetween the two groups was significant (t =- 5.14, P< 0. 01). Conclusions PEDF protein can stably express in the mice retina after rAAV2-PEDF transfetion, rAAV2-PEDF can decrease the retinal nonperfusion areas and inhibit the retinal neovascularization in OIRNV mice.     

慢病毒介导环腺苷酸反应成分结合蛋白1特异性小干扰RNA对小鼠视网膜新生血管的抑制作用

目的 观察玻璃体腔注射慢病毒介导环腺苷酸反应成分结合蛋白1（CREB1）特异性小干扰RNA（siRNA）对小鼠视网膜新生血管的抑制作用。方法 构建针对小鼠靶基因CREB1 siRNA载体,筛选并进行慢病毒包装。将140只C57BL/6J小鼠均分为正常对照组、氧诱导视网膜病变（OIR）模型组、空载体组和CREB1干扰组。正常对照组小鼠在正常空气中饲养。OIR模型组、空载体组和CREB1干扰组小鼠均于出生后7 d建立OIR模型。空载体组及CREB1干扰组小鼠于出生后5 d分别行玻璃体腔注射慢病毒 绿色荧光蛋白（GFP）空载体和CREB1-RNA干扰慢病毒载体1.0 μl。利用突破内界膜的新生血管内皮细胞核数和荧光血管造影视网膜铺片评价视网膜新生血管情况;计算小鼠视网膜新生血管和无灌注区面积;逆转录聚合酶链反应和蛋白质免疫印迹法检测小鼠视网膜CREB1 mRNA和磷酸化CREB1（P-REB1）蛋白表达,以及血管内皮生长因子（VEGF）-A、丝氨酸/苏氨酸蛋白激酶（Akt）、磷脂酰肌醇3激酶（PI3K）的mRNA和蛋白表达情况。结果 OIR模型组、空载体组突破内界膜的血管内皮细胞核计数较正常对照组明显增加,差异有统计学意义（P＜0.05）;CREB1干扰组突破内界膜的血管内皮细胞核计数较OIR模型组、空载体组明显减少,差异有统计学意义（P＜0.05）。OIR模型组、空载体组小鼠视网膜新生血管面积和无灌注区面积均较正常对照组明显增大,CREB1干扰组小鼠视网膜新生血管面积和无灌注区面积均较OIR模型组和空载体组明显缩小。4组小鼠视网膜新生血管面积和无灌注区面积比较,差异均有统计学意义（F=67.220、110.090,P＜0.05）。OIR模型组、空载体组小鼠视网膜CREB1 mRNA和P-CREB蛋白表达以及VEGF-A、Akt、PI3K mRNA和蛋白表达均较正常对照组明显增加;CREB1干扰组小鼠视网膜CREB1 mRNA和P-CREB蛋白表达以及VEGF-A、Akt、PI3K mRNA和蛋白表达较OIR模型组、空载体组明显下降。4组小鼠视网膜CREB1、VEGF-A、Akt、PI3K mRNA表达比较,差异均有统计学意义（F=6.087、5.464、6.191、8.627,P＜0.05）。4组小鼠视网膜P-CREB1、VEGF-A、Akt、PI3K 蛋白表达比较,差异也有统计学意义（F=162.944、13.861、19.710、22.827,P＜0.05）。结论 玻璃体腔注射慢病毒介导的CREB1 siRNA转染视网膜可有效抑制氧诱导小鼠视网膜新生血管的形成。     

Ras相关的C3肉毒毒素底物1的小发夹RNA干扰活性氧-核因子κB通路抑制小鼠视网膜新生血管生成

Objective To investigate the effects of knocking down Racl gene (ras-related C3 botulinum toxin substrate 1) by small hairpin RNA (shRNA) on retinal neovascularization in a mouse model of oxygen-induced retinopathy (OIR). Methods One hundred and eight 7-day-old C57BL/6J mice were divided into three groups randomly. The OIR was induced by Smith protocol in 2 groups. OIR mice received an intravitreal injection of Racl-shRNA plasmid or the nonsense plasmid in the gene-intervention group and control group respectively at the age of postnatal day 11 (P11). Non-OIR mice also received an intravitreal injection of Racl-shRNA plasmid at P11 as the blank-intervention group which lived in the normoxic environment. Retinal neovascularization was investigated on flat-mounts after fluorescence angiography at P15 and P17. Endothelial cell nuclei breaking through the internal limiting membrane were counted on pathological section at P17. The expression of Racl and NF-κB p65 subunit was measured by immuohistochemistry, Western blot, real-time polymerase chain reaction (RT-PCR) and in situ hybridization. Results Compared with the blank-control group, the level of Racl mRNA in the geneintervention group decreased obviously(t=4.5, P = 0. 001 ); the retinal non-perfusion areas, fluorescence leakage, neovascularization and the number of endothelial cell nuclei breaking through the internal limiting membrane were reduced significantly(t = 6. 521, P< 0. 001) ; the level of NF-κB p65 nuclear translocation decreased(t= 16. 008, P<0. 001)while the expression of NF-κB p65 mRNA was reduced obviously(t=3. 354, P=0. 006), which was positively correlated with the expression of Ratl mRNA (P=0. 012).Conclusion Intravitreal injection of Racl-shRNA with liposome in mice can effectively inhibit the expression of Racl, and inhibit the retinal neovascularization under relative hypoxia via blocking the ROS-NF-κB pathway.     

Ras相关的C3肉毒毒素底物1的小发夹RNA干扰活性氧-核因子κB通路抑制小鼠视网膜新生血管生成

Objective To investigate the effects of knocking down Racl gene (ras-related C3 botulinum toxin substrate 1) by small hairpin RNA (shRNA) on retinal neovascularization in a mouse model of oxygen-induced retinopathy (OIR). Methods One hundred and eight 7-day-old C57BL/6J mice were divided into three groups randomly. The OIR was induced by Smith protocol in 2 groups. OIR mice received an intravitreal injection of Racl-shRNA plasmid or the nonsense plasmid in the gene-intervention group and control group respectively at the age of postnatal day 11 (P11). Non-OIR mice also received an intravitreal injection of Racl-shRNA plasmid at P11 as the blank-intervention group which lived in the normoxic environment. Retinal neovascularization was investigated on flat-mounts after fluorescence angiography at P15 and P17. Endothelial cell nuclei breaking through the internal limiting membrane were counted on pathological section at P17. The expression of Racl and NF-κB p65 subunit was measured by immuohistochemistry, Western blot, real-time polymerase chain reaction (RT-PCR) and in situ hybridization. Results Compared with the blank-control group, the level of Racl mRNA in the geneintervention group decreased obviously(t=4.5, P = 0. 001 ); the retinal non-perfusion areas, fluorescence leakage, neovascularization and the number of endothelial cell nuclei breaking through the internal limiting membrane were reduced significantly(t = 6. 521, P< 0. 001) ; the level of NF-κB p65 nuclear translocation decreased(t= 16. 008, P<0. 001)while the expression of NF-κB p65 mRNA was reduced obviously(t=3. 354, P=0. 006), which was positively correlated with the expression of Ratl mRNA (P=0. 012).Conclusion Intravitreal injection of Racl-shRNA with liposome in mice can effectively inhibit the expression of Racl, and inhibit the retinal neovascularization under relative hypoxia via blocking the ROS-NF-κB pathway.