低氧诱导人视网膜血管内皮细胞中乙酰肝素酶和血管内皮生长因子相关性研究

目的 观察人视网膜血管内皮细胞（HREC）低氧模型中乙酰肝素酶（Hpa）、血管内皮生长因子(VEGF)和RNA 聚合酶-Ⅱ (Pol-Ⅱ)的表达变化,探讨低氧性视网膜新生血管形成中Hpa和VEGF的相关性及可能机制。方法 使用低氧模拟剂氯化钴（CoCl2）造成HREC低氧模型,分为4组,分别为正常对照组、低氧诱导组、磷酸甘露戊糖硫酸盐（PI-88）组和空白对照组。低氧诱导组为含CoCl2 100 μmol/ml的培养液培养48 h;PI-88组为含CoCl2 100 μmol/L和Hpa竞争性抑制剂PI 88 5 μg/ml的培养液干预48 h;空白对照组为等量PBS干预HREC 48 h。免疫荧光染色法观察正常对照组和低氧诱导组HREC中Hpa、VEGF以及Pol Ⅱ的表达。蛋白质免疫印迹（Western blot）检测各组间Hpa和VEGF蛋白表达变化。 结果免疫荧光染色结果显示,低氧诱导组HREC细胞质内Hpa荧光和VEGF荧光均较正常对照组增强;PI-88组细胞质内VEGF荧光较低氧诱导组减弱。低氧诱导组细胞核内Hpa较正常对照组明显增强,且分布与Pol-Ⅱ相吻合。Western blot检测结果显示,与正常对照组比较,低氧诱导组Hpa蛋白和VEGF蛋白表达均明显升高,差异有统计学意义（Hpa：F=-4.005,P＜0.05;VEGF：F=-4.063,P＜0.05）;PI-88组VEGF蛋白表达较低氧诱导组VEGF蛋白表达降低,差异有统计学意义（F=5.963,P＜0.05）。结论 低氧诱导的HREC中,Hpa的表达增高,导致VEGF的增加,促进了视网膜新生血管形成。     

Norrin基因对人视网膜微血管内皮细胞增殖和周期的影响

目的：体外培养并鉴定人视网膜微血管内皮细胞（HRCECs），并探讨Norrin基因高表达对HRCECs增殖和周期的影响。方法： 体外分离、培养并鉴定人视网膜微血管内皮细胞，抗第Ⅷ因子相关抗原抗体鉴定细胞。利用脂质体lipofectamine 2000将AP-3myc-hNorrin/pRK5质粒转染HRCECs，RT-PCR、免疫组化和Western blotting方法检测Norrin/myc的表达确定转染效率。采用MTT法测定转染后细胞的增殖能力，流式细胞术分析其细胞周期变化。结果： 所培养的细胞第Ⅷ因子相关抗原免疫组化为强阳性。AP-3myc-hNorrin/pRK5质粒转染后 24 h，RT-PCR显示实验组Norrin表达水平明显高于阴性对照组(P<0.01)。转染后48h免疫组化及Western blotting均显示实验组细胞Myc表达强于阴性对照组。MTT法显示细胞增殖高于对照组，细胞周期检测示实验组G2期细胞高于阴性对照组，P<0.01。结论：脂质体能成功转染AP-3myc-hNorrin/pRK5进入HRCECs。Norrin高表达能促进HRCECs的增殖，并促进细胞DNA合成，提示Norrin在视网膜血管生成过程中可能具有重要作用。     

急进性后极部早产儿视网膜病变的临床进程及疗效观察

目的 描述急进性后极部早产儿视网膜病变(AP-ROP)的临床进程及特征,评价视网膜光凝及冷凝对急进性后极部早产儿视网膜病变的治疗效果.方法 前瞻性、非对比性、连续性病例.2006年1月至2008年6月经检查确诊为急进性后极部ROP的患儿8例16只眼.确诊后24h内行间接眼底镜下行视网膜光凝治疗联合或不联合直视下冷凝治疗.结果 急进性后极部早产儿视网膜病变以病变大部分位于后极部1区,所有象限视网膜血管扩张迂曲,病程进展快,若不及时治疗,迅速发生视网膜漏斗状全脱离为临床特征.本组8例16只眼视网膜光凝和(或)冷凝治疗后,9只眼病变完全退化或控制,占56.2%.7只眼病情未能控制,最终发展为4b至5期视网膜病变.结论 AP-ROP进展快,预后不良,部分患儿虽经严密观察和治疗,病情仍进展.视网膜光凝和(或)冷凝治疗能控制大部分AP-ROP患儿视网膜病变的发展,挽救患儿视功能.临床上需要加强观察和随访,早发现、早诊断、早治疗是减低该病致盲率的惟一方法.     

晚期增殖性糖尿病视网膜病变的治疗价值探讨

目的探讨玻璃体视网膜手术对晚期增殖性糖尿病视网膜病变的治疗价值。方法回顾性分析VI期PDR患者33例42眼经标准的玻璃体视网膜手术治疗后的临床资料,对术前后视力,手术并发症及预后等进行重点分析。结果随访35眼,术后视力明显提高,0.05以上者从术前的6/42眼(14.29%)提高到术后的15/35眼(42.86%),其中12眼(80%)视力>0.1。一次手术视网膜复位27/35眼(77.14%),至随访末期(9月)31/35眼(88.57%)。术前伴有视盘萎缩,视网膜血管闭塞,光定位不准,新生血管性青光眼等预后较差,但术后视力仍有11/16(68.8%)眼有不同程度的提高,6/16(37.5%)眼视力≥FC。结论玻璃体视网膜手术可以部分改善晚期PDR患者的视功能,即便是合并严重缺血的病例仍有一定的治疗价值。     

色素上皮衍生因子在氧诱导血管增生性视网膜病变小鼠中的表达

目的探讨氧诱导血管增生性视网膜病变小鼠视网膜中色素上皮衍生因子(PEDF)mRNA及蛋白的表达变化及意义。方法80只出生后2 d的C 57/BL 6小鼠随机分为模型组(64只)和对照组(16只),出生后第7天模型组小鼠和母鼠一起放入氧气含量为75%的饲养箱中饲养,出生后第12天回到正常大气环境中饲养;对照组小鼠始终在正常大气环境中饲养。出生后第7、12、17天时,模型组及对照组在每个时间点分别取小鼠4只,荧光素心脏灌注后行视网膜铺片,荧光显微镜下观察血管分布和形态;提取模型组小鼠出生后第7、8、10、12、12.5、13、14、15、17、19天时间点的视网膜总RNA,通过逆转录-聚合酶链式反应(RT-PCR)检测PEDF、血管内皮生长因子(VEGF)、低氧诱导因子(H IF)-1αmRNA的表达变化;分别取模型组和对照组出生后第17天时的小鼠眼球,冰冻切片后通过免疫组织化学染色检测PEDF、VEGF、H IF-1α蛋白在视网膜组织中的表达变化。结果模型组小鼠在出生后第17天时视网膜有大量新生血管形成,视盘周围见大范围无灌注区;PEDF mRNA表达在缺氧24 h后开始下调,72 h达到下降高峰,随后逐渐恢复正常水平;VEGF、H IF-1αmRNA则在高氧环境下表达下调,缺氧状态下迅速升高,于缺氧48 h时到达高峰,然后缓慢下降;PEDF/VEGF、PEDF/H IF-1α比值在缺氧状态下显著降低。出生后第17天时,模型组小鼠视网膜中PEDF蛋白表达显著弱于对照组小鼠;而VEGF、H IF-1α蛋白表达则是模型组显著强于对照组。结论PEDF mRNA及蛋白在氧诱导血管增生性视网膜病变小鼠视网膜中明显下降,其趋势与VEGF和H IF-1α的变化相反,PEDF/VEGF、PEDF/H IF-1α表达平衡失调,可能是缺氧状态下视网膜新生血管生成的重要原因。     

糖尿病性视神经病变的临床分析

目的 观察糖尿病性视神经病变的临床表现、眼底血管造影和视野特征.方法 对173例321眼0～Ⅴ期糖尿病视网膜病变患者的临床表现、视力、视野、荧光造影等临床资料进行分析.结果 321眼中有视神经病变者共155眼,占48.3%.临床表现多样,轻者无任何症状,重者视力下降明显.眼底检查可有视盘水肿、色淡、充血等多种改变.荧光造影中主要表现为视盘新生血管40眼,占25.8%、视盘充盈缺损21眼,占13.5%、视盘水肿45眼,占29.0%及视盘晚期染色49眼,占31.6%.0期DR患者中,25.9%视神经有异常改变.Ⅰ～Ⅴ期DR患者视神经异常率分别为25.0%,37.5%,43.0%,65.1%,85.1%.结论 糖尿病性视神经病变发生率高,临床表现多种多样.糖尿病视网膜病变越严重,发生糖尿病视神经病变的可能性越大,但两者不平行.荧光造影检查对糖尿病视神经病变的早期诊断有重要意义.糖尿病性视神经病变是影响视力和预后的重要因素,应引起眼科医生的高度重视.     

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.     

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.     

周边视网膜变性广角多模影像研究进展

周边视网膜变性是眼科临床常见的病变,不同类型的变性影响不同的视网膜层次,可能对视力造成威胁。尽管现代眼底成像技术被应用于研究其病理生理机制,由于其所处的特殊部位,影像学检查困难,因此发病机制仍不清楚。本文总结了有关周边视网膜变性的多种广角成像技术的影像特征,包括超广角眼底成像、广角频域光学相干断层扫描、光学相干断层扫描血管成像、荧光素眼底血管造影等,及其发病机制或病理特点的新观点,为临床诊疗提供新的思路。由于样本量非常少,且缺乏前瞻性、长期的多模态影像的观察研究,因而目前仍无法全面评价不同类型病变的进展性及危险性。期望在更广的范围内应用多模态广角成像技术对此类疾病进行研究,指导临床干预决策。