可溶性fms-样酪氨酸激酶受体-1不同基因片段对视网膜新生血管的抑制作用

Objective To investigate the inhibitory effects of fms-like typrosine kinase receptor sFh-1 on retinal neovascularization (RNV). Methods Recombinant lentivirus sFh-1 ( 2-3 ) and sFh-1 ( 2-4 )expressing the sFh-1 (2-3) and (2-4) immunoglobulin-like regions of sFh-1 were constructed. 96 seven-dayold C57/6J mice were randomly divided into 4 groups with 24 mice in each group. Group 1 : normal control;group 2: experimental control; group 3: sFlt-1(2-3); group 4: sFlt-1(2-4). The mice in group 2-4 were exposed to hyperoxia with (75±2)% O2 for 5 days and then returned to normoxia with 21% O2 ; the mice     

可溶性fms-样酪氨酸激酶受体-1不同基因片段对视网膜新生血管的抑制作用

Objective To investigate the inhibitory effects of fms-like typrosine kinase receptor sFh-1 on retinal neovascularization (RNV). Methods Recombinant lentivirus sFh-1 ( 2-3 ) and sFh-1 ( 2-4 )expressing the sFh-1 (2-3) and (2-4) immunoglobulin-like regions of sFh-1 were constructed. 96 seven-dayold C57/6J mice were randomly divided into 4 groups with 24 mice in each group. Group 1 : normal control;group 2: experimental control; group 3: sFlt-1(2-3); group 4: sFlt-1(2-4). The mice in group 2-4 were exposed to hyperoxia with (75±2)% O2 for 5 days and then returned to normoxia with 21% O2 ; the mice     

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.     

可溶性VEGF受体1在视网膜新生血管疾病中的研究进展

视网膜新生血管性疾病的共同特征是病理性新生血管形成。目前研究的内源性视网膜新生血管因子最主要的是VEGF。可溶性VEGF受体1(sFlt-1)是VEGFR-1的mRNA胞外区剪接形成的可溶性形式,只编码胞外区,缺乏细胞内酪氨酸激酶区域,所以其仅具有与配体结合的能力,而无信号转导能力,从而阻止新生血管的形成。sFlt-1作为近年来的研究热点,有可能成为治疗该类疾病的新的基因治疗方法。本文就sFlt-1在视网膜新生血管疾病治疗中的作用机制及研究进展做一综述。     

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.     

羧甲基葡聚糖磁性纳米颗粒与脂质体介导人可溶性fms-样酪氨酸激酶受体-1的转染比较

目的 比较羧甲基化葡聚糖(CMD)磁性纳米颗粒与脂质体LipofectamineTM2000对人可溶性fms-样酪氨酸激酶受体-1(sFlt-1)第2～4区基因片段的转染率.方法 构建真核表达质粒编码增强型绿色荧光蛋白质粒(pcDNA3.1-EGFP)/sFlt-1(2～4),采用酶切、电泳及基因测序鉴定.将实验分为磁颗粒组、脂质体组和未转染对照组进行.转染后24、48 h,倒置相差荧光显微镜下观察细胞绿色荧光分布;流式细胞仪检测细胞绿色荧光表达率;逆转录聚合酶链反应(RT-PCR)法和免疫蛋白印迹(Western blot)法检测sFlt-1(2-4)mRNA和蛋白表达;噻唑蓝(MTT)比色法观测细胞生长情况,计算各组细胞相对增长率(RGR);Hoeehst细胞核染色法观察各组细胞凋亡情况.结果 重组质粒pcDNA3.1/sFlt-1(2～4)酶切产物在琼脂糖凝胶电泳时出现大小为915碱基对的条带.流式细胞仪检测发现,磁颗粒组平均转染率为45%,脂质体组平均转染率21%;二者比较,差异有统计学意义(t=2.541,P<0.05).RT-PCR和Western blot观察发现,转染后48 h磁颗粒组细胞sFlt-1(2～4)mRNA和蛋白表达均明显高于脂质体组(t=2.454,2.398;P值均<0.05).转染后24、48 h,磁颗粒组RGR与未转染对照组间差异无统计学意义(t=1.436,P>0.05),脂质体组RGR与未转染对照组及磁颗粒组间差异均有统计学意义(t=2.412,2.545,P值均<0.05);磁颗粒组细胞凋亡率与未转染对照组间差异无统计学意义(t=1.436,P>0.05),与脂质体组间差异有统计学意义(t=2.236,P<0.05).结论 CMD磁性颗粒较脂质体LipofeetamineTM2000可获得更高的sFlt-1(2～4)基因片段转染率.     

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.     

两种fms-样络氨酸激酶受体-1基因片段对磷脂酰肌醇-3激酶/蛋白酶B之间信号通路的影响

目的 观察可溶性fms-样络氨酸激酶受体-1(sFlt-1)基因胞外第2～3区和2～4区对缺氧、高糖环境下视网膜血管通透性及磷脂酰肌醇-3激酶/蛋白酶B(PI3K/Akt)之间信号通路的影响.方法 构建真核表达质粒编码增强型绿色荧光蛋白质粒(pcDNA3.1-EGFP)/s Flt-1(2～3)和pcDNA3.1-EGFP/s Flt-1(2～4),采用酶切、电泳及基因测序鉴定.将实验分为正常对照组、缺氧组和高糖组进行.以羧甲基化匍聚糖(CMD)纳米磁颗粒为基因载体,分别携带两种重组质粒转染缺氧、高糖条件下培养的人脐静脉血管内皮细胞(HUVEC).采用分光光度计检测各组兔视网膜血管对伊凡思蓝(EB)染料的渗漏量.逆转录聚合酶链反应(RT-PCR)和免疫蛋白印迹(Western blot)法分别检测各组细胞PI3K/Akt信号通路下游特异性磷酸化激酶Akt(p-Akt)mRNA和蛋白的表达.结果 缺氧组和高糖组注射转染细胞上清液后,视网膜血管EB渗漏量较正常对照组明显增加(t=2.476,2.515;P值均＜0.01).缺氧组和高糖组转染了pcDNA3.1-EGFP/sFlt-1(2～3)和pcDNA3.1-EGFP/sFlt-1(2～4)后,视网膜血管EB渗漏量较未转染明显降低(t=2.598,2.679,2.386,2.732;P值均＜＜0.01).缺氧组、高糖组HUVEC p-Akt mRNA表达较正常对照组明显上调(t=2.612,2.545;P值均＜0.01),转染pcDNA3.1-EGFP/sFlt-1(2～3)和pcDNA3.1-EGFP/sFlt-1(2～4)后明显下调(t=2.512,2.189,2.372,2.687;P值均＜0.01).缺氧组和高糖组HUVEC p-Akt蛋白表达较正常对照组明显上调(t=2.376,2.712;P值均＜0.01),转染pcDNA3.1-EGFP/sFlt-1(2～3)和pcDNA3.1-EGFP/sFlt-1(2～4)后明显下调(t=2.259,2.391,2.399,2.413;P值均＜0.01).结论 sFlt-1受体胞外第2～3区和2～4区均可抑制缺氧、高糖环境下的视网膜血管通透性增高和PI3K/Akt通路的信号传导.     

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

目的 观察重组腺相关病毒-色素上皮衍生因子(rAAV2-PEDF)对氧诱导小鼠视网膜新生血管(RNV)的作用.方法 选择3日龄C57/BL6小鼠22只,左眼为实验服,右眼为对照眼,微量注射器玻璃体腔分别注射rAAV2-PEDF和rAAV2-绿色荧光蛋白1μl.注射后立即将小鼠放入氧箱,建立氧诱导血管增生性视网膜病变模型.取13日龄小鼠4只提取视网膜总蛋白,蛋白质免疫印迹法(Western blot)检测色素上皮衍生因子(PEDF)蛋白表达.17日龄小鼠12只,荧光素心脏灌注视网膜铺片观察血管形态和分布.17日龄小鼠6只,视网膜冰冻切片外源凝集素标记后染色观察血管形态和分布.Image-Pro Plus5.1软件测量分析无荧光素灌注区和RNV的绝对面积和相对面积.结果 实验眼PEDF蛋白表达显著高于对照眼.视网膜铺片定量结果显示,实验眼和对照跟绝对无灌注区面积分别为(0.96±0.22)、(1.96±0.34)mm2,差异有统计学意义(t=-8.554,P<0.01);相对无灌注区面积分别为(8.64±1.52)%、(17.27±2.98)%,差异有统计学意义(t=-8.97,P<0.01).实验眼和对照眼绝对新生血管面积分别为(0.37±0.11)、(1.26±0.38)mm2,差异有统计学意义(t=-7.8,P<0.01);相对新生血管面积分别为(3.96±0.66)%、(11.45±2.06)%,差异有统计学意义(t=-8.51,P<0.01).外源凝集素标记定量结果显示,实验眼和对照眼RNV面积分别为(0.11±0.003)、(0.41 4-0.02)mm2,差异有统计学意义(t=-5.14,P<0.01).结论 rAAV2-PEDF可成功转染小鼠视网膜组织并稳定表达PEDF蛋白,不仅可以减少氧诱导血管增生性视网膜病变小鼠视网膜无灌注面积,而且可显著抑制RNV生成.     

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。