缺氧诱导因子1α对早期糖尿病视网膜病变状态下人粒细胞系白血病细胞表面黏附分子CD18表达的调节

目的　观察在早期糖尿病视网膜病变(DR)状态下,缺氧诱导因子1α（HIF-1α）对细胞表面黏附分子CD18表达以及白细胞和血管内皮细胞黏附的影响。方法　收集早期DR患者及年龄匹配的健康人外周血血清,用于体外培养人粒细胞系白血病细胞（HL60）和恒河猴视网膜血管内皮细胞系细胞（RF/6A）。分为糖尿病血清培养组（B组）、糖尿病＋HIF 1α反义寡核苷酸组（ASODN）（C组）、糖尿病＋HIF 1α 正义寡核苷酸组（SODN）（D组）,健康人血清培养细胞为正常对照组（A组）。以流式细胞仪和Real-time PCR分别检测HL60细胞表面CD18蛋白阳性细胞比例和CD18 mRNA的表达水平,以虎红染色法观察HL60细胞和RF/6A细胞的黏附率。结果　A、B、C、D组CD18阳性细胞比例分别为17.06±6.01、42.23±2.60、25.33±3.05、32.40±10.57,各组之间差异有统计学意义（F=36.47,P＜0.001）。和A组比较,B、C、D组CD18 mRNA水平分别是A组的21.05±2.07、2.23±0.96、25.07±2.27倍,各组之间差异有统计学意义（F＝180.34,P＜0.001）。A、B、C、D组细胞黏附率分别为0.06±0.002、0.09±0.10、0.05±0.007、0.07±0.01,各组之间差异有统计学意义（F=13.06,P=0.002）。结论　在体外培养条件下,糖尿病血清可以促进HL60细胞表达CD18蛋白和CD18mRNA,促进HL60细胞和血管内皮细胞的黏附,HIF-1α表达被抑制后,这种促进作用减弱。HIF-1α对糖尿病状态下细胞表面黏附分子CD18表达及白细胞和血管内皮细胞之间的黏附具有调节作用。      

顺铂对视网膜母细胞瘤细胞B7-H1表达的影响

　　目的　观察顺铂对视网膜母细胞瘤（RB）细胞B7-H1表达的影响,并探讨其作用机制。方法　分别采用浓度为0.375、0.750、1.500、3.000、6.000 μg/ml顺铂处理人RB细胞HXO-Rb₄₄细胞,作为不同浓度实验组;以RPMI 1640细胞培养液处理HXO-Rb44细胞作为空白对照组。采用逆转录-聚合酶链反应（RT-PCR）和荧光定量聚合酶链反应（PCR）检测HXO-Rb₄₄细胞B7-H1 mRNA的表达;免疫荧光染色和流式细胞术检测HXO-Rb₄₄细胞B7-H1蛋白表达。采用1.500 μg/ml顺铂处理HXO-Rb₄₄细胞0、15、30、60、120 min,蛋白免疫印迹（Western blot）检测细胞外信号调节激酶1/2（ERK1/2）的磷酸化水平。结果　0.375、0.750、1.500、3.000、6.000 μg/ml组HXO-Rb₄₄细胞B7-H1 mRNA表达均较空白对照组高,差异有统计学意义（F=395.478,P=0.000）。0.375、0.750、1.500、3.000、6.000 μg/ml组HXO-Rb₄₄细胞B7-H1蛋白表达均较空白对照组高,差异有统计学意义（F=112.03,P=0.000）。 Western blot 检测显示,1.500 μg/ml顺铂激活HXO-Rb₄₄细胞ERK1/2蛋白的磷酸化水平;随时间延长,其磷酸化水平逐渐增高,至30 min达高峰,以后又逐步下降。结论　顺铂能促进RB细胞B7-H1 mRNA和蛋白的表达;ERK1/2信号通路可能参与了这一过程。      

糖尿病小鼠眼opticin蛋白表达的研究

目的　观察发生糖尿病与未发病非肥胖性糖尿病（NOD）小鼠玻璃体内opticin蛋白表达差异。方法　建立糖尿病NOD小鼠模型作为实验组,选用同龄NOD小鼠作为对照。颈椎脱臼法处死两组所有小鼠,摘除眼球,钝性分离玻璃体、视网膜、巩膜。采用蛋白质免疫印迹（Wesrern blot）和实时定量逆转录聚合酶链反应（RT-PCR）对比分析实验组和对照组玻璃体、视网膜、巩膜中opticin蛋白及其相关基因（OPTC）mRNA的表达。结果　Western blot检测显示,实验组和对照组玻璃体、视网膜及巩膜中均可见opticin蛋白表达条带,主要位于相对分子质量60×10³处。实验组玻璃体、视网膜中opticin蛋白较对照组显著降低(t=4.42,4.58;P=0.002,0.002),巩膜中opticin蛋白差异无统计学意义（t=0.27,P=0.794）。RT-PCR检测显示,OPTC mRNA表达分布规律为玻璃体、视网膜、巩膜依次递减。实验组玻璃体、视网膜中OPTC mRNA表达较对照组显著降低(t=3.30,2.48;P=0.01,0.04);巩膜中OPTC mRNA表达差异无统计学意义（t=0.27,P=0.80）。结论　糖尿病NOD小鼠玻璃体、视网膜中opticin蛋白表达较未发病NOD小鼠受抑制。      

在体电穿孔辅助基因转染视网膜色素上皮细胞层和光感受器细胞层的实验研究

目的　探讨在体电穿孔辅助基因转染视网膜色素上皮（RPE）细胞层和光感受器（PR）细胞层的可行性。方法　147只健康雄性Spragne-Dawley(SD)大鼠根据电穿孔刺激模式电压值分为5、10、15、20、25、30、35 V组,右眼视网膜下腔注射增强型绿色荧光蛋白（EGFP）真核表达质粒pEGFP N1为实验眼,左眼注射TE Buffer 为对照眼。各组根据不同转染方向再分为RPE和PR两个亚组。各组除电压不同外,其余参数均为脉宽99 ms,脉冲间期0.5 s,连续5个脉冲。将带有负电荷的质粒在电场作用下,拟转染到RPE细胞层,反向置电极,拟转染到PR细胞层。转染后7 d 取各组视网膜铺片,荧光显微镜下观察EGFP表达强弱、范围及荧光细胞计数分析;采用蛋白质免疫印迹法（Western blot）、实时逆转录聚合酶链反应（RT-PCR）半定量观察EGFP蛋白和mRNA的表达。结果　转染后7 d,荧光显微镜观察发现,各组RPE亚组对照眼RPE细胞层内均未见特异性荧光表达,实验眼可见绿色荧光表达;各组RPE亚组对照眼视网膜铺片均未见荧光表达,实验眼视网膜铺片均可见转染了EGFP的RPE细胞。Western blot检测显示,随电压增加,EGFP蛋白与β-肌动蛋白条带灰度相对比值呈上升趋势。RT-PCR检测显示,各组均产生阳性扩增条带,随电压增高,EGFP mRNA与GADPH mRNA扩增条带灰度相对比值逐渐增高。结论　在体电穿孔方法可以有效辅助基因转染大鼠RPE细胞层。      

糖尿病大鼠早期视网膜小胶质细胞活化与神经节细胞损害的关系

目的　观察糖尿病早期视网膜小胶质细胞的活化特征与视网膜神经节细胞（RGC）损害的关系。方法　20只成年雄性Sprague-Dawley（SD）大鼠,采用链脲佐菌霉素腹腔注射方法制作糖尿病动物模型,分为糖尿病1、3个月组及相应正常对照组,每组5只大鼠。对所有大鼠行上丘定位注射逆行标记RGC,分别用免疫组织化学法标记视网膜铺片、冰冻切片小胶质细胞和RGC,共聚焦显微镜下观察小胶质细胞细胞形态及分布特征。结果　糖尿病组视网膜铺片小胶质细胞胞体增粗,形态不规则。与对照组相比,糖尿病3个月组RGC层发生吞噬的小胶质细胞密度显著增加（t=3.83,P＜0.01）。与对照组相比,糖尿病大鼠1、3月个组RGC层小胶质细胞平均密度均显著增加（t=2.71,4.22;P＜0.05）;糖尿病大鼠3个月组RGC层小胶质细胞平均密度较糖尿病1个月组显著增加（t=7.45,P＜0.0001）。糖尿病早期小胶质细胞与RGC数量之间存在相关关系（r=0.9,P＜0.05）。结论　糖尿病早期小胶质细胞活化与RGC损伤关系密切。     

蓝光照射后视网膜αA-和αB-晶体蛋白表达变化

目的　观察蓝光照射后大鼠视网膜中αA和αB晶体蛋白的表达。方法　40只雌性Wistar大鼠随机分为正常对照组,蓝光照射6、12、24 h组共4组,每组10只。正常对照组不给予任何处理,蓝光照射6、12、24 h组分别给予蓝光照射6、12、24 h后给予12 h暗适应,麻醉后摘取眼球。采用免疫组织化学和蛋白质免疫印迹(Western blot)法检测视网膜中αA和αB晶体蛋白表达。结果　免疫组织化学染色检测显示,正常对照组以及蓝光照射6、12、24 h组视网膜αA 晶体蛋白吸光度［A,旧称光密度（OD）］值分别为1.405 73±0.707 48、4.317 51±0.412 97、7.397 08±1.947 90、9.634 32±2.377 61,组间比较差异均有统计学意义（F=24.569,P＜0.001）。正常对照组以及蓝光照射6、12、24 h组视网膜αB 晶体蛋白A值分别为0.129 36±0.033 93、0.507 17±0.117 55、7.345 43±2.292 97、4.042 26±3.890 23,组间比较差异均有统计学意义（F=40.102,P＜0.001）。Western blot 检测发现,蓝光照射6、12、24 h后视网膜αA-及αB-晶体蛋白表达明显高于正常对照组。结论 蓝光可诱导大鼠视网膜αA-晶体蛋白和αB-晶体蛋白表达上调。      

磷脂酰肌醇-3激酶信号通路在缺氧诱导的人视网膜色素上皮细胞VEGF表达中的作用

目的:探讨磷脂酰肌醇-3激酶(phosphatidylinositol3-ki-nase,PI3K)信号转导通路对缺氧诱导的视网膜色素上皮(retinalpigmentepithelium,RPE)细胞表达VEGF的影响。方法:利用CoCl2建立培养的人RPE细胞缺氧模型,分为单纯缺氧组和30μmol/LPI3K特异性阻断剂LY294002阻断处理组,在缺氧条件下分别培养0,1,4,8,12和24h。细胞免疫荧光法检测磷酸化PI3K表达水平;酶联免疫吸附试验(enzymelinkedimmunosorbentassay,ELISA)检测RPE细胞上清中VEGF的含量。结果:缺氧刺激1,4,8,12和24h,RPE细胞膜上PI3K磷酸化表达水平逐渐增高(P<0.05);随缺氧时间延长,RPE细胞上清液中VEGF含量逐渐增加(P<0.05);LY294002处理组VEGF含量显著低于对照组(P<0.05)。结论:PI3K信号转导通路参与了缺氧引起的人RPE细胞VEGF表达的调控。     

αA-晶体蛋白在2型糖尿病大鼠神经视网膜的异常表达

目的　用蛋白质组学方法观察αA-晶体蛋白在早期2型糖尿病大鼠神经视网膜的异常表达。方法　28 只成年雄性Sprague-Dawley(SD)大鼠随机分为正常对照组和糖尿病组,每组14只。糖尿病组大鼠采用高脂饮食饲养联合小剂量链脲佐菌素(STZ)注射诱导2型糖尿病模型(T2DM),以随机血糖持续高于16.7 mmol/L为模型建立成功标准。对照组用常规饲料喂养,腹腔注射相同剂量枸橼酸钠缓冲液。成模56 d 后断颈处死所有大鼠,剥离留存神经视网膜组织。从对照组和糖尿病组各取3只大鼠的神经视网膜组织,提取总蛋白,用二维凝胶电泳（2-DE）方法分别进行分离。应用基质辅助激光解吸电离飞行时间串联质谱 (MALDI-TOF-MS/MS) 和肽质量指纹图谱 (PMF) 技术对部分差异表达蛋白进行鉴定,并用蛋白免疫印迹（Western blot）和间接免疫荧光（IMF）方法对在糖尿病组表达上调的αA-晶体蛋白进行验证。结果　平均每块凝胶检测到的蛋白斑点,对照组为（3122±37）,糖尿病组为（2702±21）个。二者比较,表达水平差异有统计学意义的斑点约150个（t值均＞2.57,P值均＜0.05）。在糖尿病组表达上调68个,表达下调82个。质谱分析和数据库检索鉴定出 20个差异表达蛋白斑点。其中二维凝胶中2369和1048斑点在糖尿病组呈现高表达。经质谱PMF鉴定为αA-晶体蛋白。Western blot 检测结果显示,αA-晶体蛋白在糖尿病组大鼠神经视网膜表达明显增高。IMF 结果显示 αA-晶体蛋白在糖尿病组高表达,阳性表达主要位于视网膜的内外核层,在细胞的核周细胞浆区域有强荧光聚集。结论　αA-晶体蛋白在早期2型糖尿病大鼠神经视网膜表达增高。      

抗血管内皮生长因子单克隆抗体ranibizumab治疗渗出型老年性黄斑变性方案的探讨

目的 观察抗血管内皮生长因子（VEGF）单克隆抗体ranibizumab（商品名Lucentis）玻璃体腔注射治疗渗出型老年性黄斑变性（AMD）的临床疗效。方法 回顾分析临床确诊为渗出型AMD并接受玻璃体腔注射ranibizumab治疗的46例患者52只眼的临床资料。患者均进行了糖尿病早期治疗研究（ETDRS）视力表、检眼镜、荧光素眼底血管造影（FFA）和（或）吲哚青绿血管造影（ICGA）以及光相干断层扫描（OCT）检查。确诊渗出型AMD后,采用10 mg/ml的 ranibizumab 0.05 ml玻璃体腔注射治疗。每一个月注射1次,连续注射3次,随后根据每一个月的检查情况决定是否进行再次注射。52只眼共注射214次,每一只眼注射2~6次,平均注射4.12次。随访观察12个月。对比分析治疗前后视力、视网膜厚度及脉络膜新生血管（CNV）病灶渗漏变化情况。结果 治疗后12个月,52只眼ETDRS视力表的平均字母数是37.8个,较治疗前提高11.4个（t=-3.475,P＜0.01）。CNV病灶渗漏完全停止11只眼,占21.2%;渗漏范围明显减少34只眼,占65.4%;渗漏无明显变化者4只眼,占7.7%;病灶增大2只眼,占3.8%;新病灶出现1只眼,占1.9%。OCT检查显示,视网膜厚度平均值为187.50 μm ,较治疗前下降122.80 μm（t=4.593,P＜0.01）。结论 按治疗方案进行的ranibizumab玻璃体腔注射治疗渗出型AMD可使视力提高、视网膜水肿明显减轻、CNV病灶渗漏停止或减少。      

Focal adhesion kinase signaling pathway participates in the formation of choroidal neovascularization and regulates the proliferation and migration of choroidal microvascular endothelial cells by acting through HIF-1 and VEGF expression in RPE cells

Choroidal neovascularization (CNV) is one of the most frequent causes of severe and progressive vision loss, while its pathogenesis is still poorly understood. Focal adhesion kinase (FAK), a non-receptor tyrosine kinase, plays a crucial role in linking signals initiated by both the extracellular matrix (ECM) and soluble signaling factors and controls essential cellular processes. Extensive evidence has shown that FAK is activated in angiogenic response. This study aims to investigate the effect of FAK on CNV formation. The Brown-Norway (BN) rats underwent laser rupture of Bruch's membrane to induce CNV and were then killed at 1, 3, 7, and 14 days following laser injury. Immunofluorescence and Western blot were processed to detect FAK protein. Retinal pigment epithelial (RPE) cells were cultured under hypoxia and RNA interference (RNAi) technique was used to knock down the FAK gene in RPE cells. Expression of hypoxia inducible factor-1 (HIF-1α) and vascular endothelial growth factor (VEGF) in RPE cells were investigated by RT-PCR and Western blot. Two kinds of coculture models were used to observe the effects of specific blockade of FAK in RPE cells on the proliferation and migration of choroidal microvascular endothelial cells (CECs), respectively. FAK was highly expressed in the rat RPE-choroid tissue after photocoagulation. In vitro experiment showed that FAK was involved in hypoxia signaling in cultured RPE cells. The absence of FAK effectively reduced the expression of hypoxia-induced HIF-1α and VEGF in RPE cells, resulting in the inhibition of proliferation and migration of CECs. Our results suggest that FAK pathway activation plays a role in the development of CNV, and regulates the proliferation and migration of CECs by acting through HIF-1 and then up-regulating the expression of the angiogenic factor VEGF in RPE cells. It is reasonable to propose that FAK siRNA will potentially provides a means to attenuate the strong stimuli for neovascularization in CNV-dependent disorders, which could present a therapeutically relevant strategy for the inhibition of CNV.     

Inhibition of proliferation,migration and tube formation of choroidal microvascular endothelial cells by targeting HIF-1α with short hairpin RNA-expressing plasmid DNA in human RPE cells in a coculture system

BACKGROUND: Retinal pigment epithelial (RPE) cells and choroidal microvascular endothelial cells (CECs) are the main cells involved in choroidal neovascularization (CNV), and hypoxia plays an important role in CNV formation via hypoxia inducible factor 1 (HIF-1). Our aim was to evaluate the role of HIF-1 in human RPE cells with regard to proliferation, migration and tube formation of CECs under hypoxia. METHODS: RPE cells were cultured under chemical hypoxia induced by 200 muM CoCl(2), and RNA interference (RNAi) technique was used to knock down HIF-1alpha gene in RPE cells. mRNA and protein expression of HIF-1alpha and VEGF in RPE cells were investigated by real-time RT-PCR and Western blot. Three kinds of coculture models were used to observe the effects of RPE cells transfected by short hairpin RNA (shRNA)-expressing plasmid DNA (pDNA) (pshHIF-1alpha) on the proliferation, migration and tube formation of CECs respectively. RESULTS: Transfection of shRNA-expressing pDNA targeting HIF-1alpha to RPE cells resulted in the knock down of HIF-1alpha gene and reduction of the corresponding mRNA and protein of HIF-1alpha and VEGF under hypoxia. Consequently, the proliferation, migration and tube formation of CECs were significantly inhibited by the knocked-down RPE cells compared with the control in the coculture system. The proliferation rates of CECs decreased by 40.2%, 36.6% and 36.8% on days 3, 4 and 5 respectively. Migration reduced by 49.6% at 5 h, and tube formation decreased by 40.4% at 48 h. CONCLUSION: RNAi of HIF-1alpha in RPE cells can inhibit angiogenesis in vitro and provide a possible strategy for treatment of choroidal neovascularization diseases by targeting HIF-1alpha.     

Impacts of hypoxia-inducible factor-1 knockout in the retinal pigment epithelium on choroidal neovascularization

Purpose. Hypoxia-inducible factor (HIF)-1 is a key oxygen sensor and is believed to play an important role in neovascularization (NV). The purpose of this study is to determine the role of retinal pigment epithelium (RPE)-derived HIF-1α on ocular NV. Methods. Conditional HIF-1α knockout (KO) mice were generated by crossing transgenic mice expressing Cre in the RPE with HIF-1α floxed mice, confirmed by immunohistochemistry, Western blot analysis, and fundus fluorescein angiography. The mice were used for the oxygen-induced retinopathy (OIR) and laser-induced choroidal neovascularization (CNV) models. Results. HIF-1α levels were significantly decreased in the RPE layer of ocular sections and in primary RPE cells from the HIF-1α KO mice. Under normal conditions, the HIF-1α KO mice exhibited no apparent abnormalities in retinal histology or visual function as shown by light microscopy and electroretinogram recording, respectively. The HIF-1α KO mice with OIR showed no significant difference from the wild-type (WT) mice in retinal levels of HIF-1α and VEGF as well as in the number of preretinal neovascular cells. In the laser-induced CNV model, however, the disruption of HIF-1α in the RPE attenuated the over expression of VEGF and the intercellular adhesion molecule 1 (ICAM-1), and reduced vascular leakage and CNV area. Conclusions. RPE-derived HIF-1α plays a key role in CNV, but not in ischemia-induced retinal NV.     

Rac1 activates HIF-1 in retinal pigment epithelium cells under hypoxia

Background  Upregulation of vascular endothelial growth factor (VEGF) in hypoxic retinal pigment epithelium (RPE) cells, mediated by hypoxia-inducible factor-1 (HIF-1) is responsible for choroidal neovascularization (CNV). HIF-1α is the inducible subunit of HIF-1, but the underlying mechanisms by which RPE cells sense a decrease in oxygen concentration and transduce this signal to HIF-1α are largely unknown. Rho family small GTPase Rac1, as a potential intermediate, possibly plays a pivotal role in activating HIF-1α in RPE cells under hypoxia. Aims  To further define Rac1 playing an essential role in the induction of HIF-1α expression in RPE cells under hypoxia. Methods  In this study, we examined the expression of HIF-1α and Rac1 in human RPE cells under hypoxia for 0, 1, 2, 4, 8, 12 and 24 h by RT-PCR and Western blot. To elucidate whether Rac1 is responsible for activating the expression of hypoxia-induced HIF-1α, human RPE cells were treated with Rac1 inhibitor NSC23766 under hypoxia for 0, 1, 2, 4, 8, 12 and 24 h, and expression of HIF-1α and Rac1 measured by RT-PCR and Western blot. Results  The mRNA expression of HIF-1α and Rac1 in RPE cells significantly increased in a time-dependent manner, reaching the maximum at 4 h, and thereafter slowly declined. HIF-1α protein induction in human RPE cells was found after 1 h of hypoxia, reaching the maximum at 8 h, and then slowly declined. In response to hypoxia, the levels of Rac1 protein significantly increased, reaching the maximum at 4 h, and then slowly declined. After treatment with NSC23766, both HIF-1α and Rac1 expression were significantly inhibited in hypoxic RPE cells. Conclusions  Rac1 is crucial to activate HIF-1 in RPE cells under hypoxia, which may be a novel target other than VEGF and HIF-1 in developing CNV inhibitors.     

JAK/STAT pathway mediates retinal ganglion cell survival after acute ocular hypertension but not under normal conditions

Intraocular pressure (IOP) elevation is an important cause of glaucoma. Animal models of ocular hypertension have been widely used to mimic glaucoma to investigate the mechanisms underlying retinal ganglion cell (RGC) death and search for possible cure. The aim of the present study was to examine the role of JAK/STAT pathway in RGC viability in normal condition or after acute IOP elevation. Retinal explants obtained from intact or IOP-elevated eyes were firstly used to examine the effect of the JAK/STAT pathway inhibitors, AG490 and Jak Inhibitor I, on RGC viability in vitro. The role of this signal pathway was further investigated and confirmed in vivo. AG490 and Jak Inhibitor I were applied into the left eye on days 3, 9, and 15 post 2-h IOP elevation at 110mmHg. Fluorescence dye Fluorogold was used to retrogradely label surviving RGCs. Because macrophage recruitment was seen in the IOP-elevated eyes after inhibition of this pathway, clodronate liposomes were used to remove phagocytic cells in the eye and examine the role of JAK/STAT pathway in RGC survival independent of macrophages. Activities and location of JAK/STAT pathway in the retina were examined using Western blotting and immunohistochemistry. We found that inhibition of JAK/STAT pathway did not affect RGC survival in the retinal explants derived from intact eye but caused RGC death in the retinal explants that were derived from IOP-elevated eye. Importantly, the detrimental effect of JAK/STAT pathway inhibition on RGC survival was also observed in vivo following acute IOP elevation, but not in intact eye. In addition, both in vitro and in vivo experiments confirmed a detrimental action of phagocytic cells following acute IOP elevation and the pathway inhibition. Compatible with what were observed in vivo, Western blotting and immunohistochemistry showed that JAK/STAT activities were not present in intact retina, but acute IOP elevation activated JAK/STAT pathway in the retina, in the regions of inner nuclear layer and ganglion cell layer, including RGCs. The IOP elevation-induced JAK/STAT activities were effectively abolished by intravitreal application of AG490. This study thus shows that (1) acute IOP elevation activates JAK/STAT pathway in RGCs, and (2) JAK/STAT pathway mediates RGC survival following IOP elevation but not under normal condition.     

shRNA抑制人RPE细胞中HIF-1α表达下调后IGF-1对VEGF表达的影响

背景 增生性玻璃体视网膜疾病(PVD)是一组眼底视网膜血管性疾病,主要由视网膜色素上皮( RPE)细胞增生所致,胰岛素样生长因子-1(IGF-1)和血管内皮生长因子(VEGF)与RPE细胞的异常增生和病理性新生血管生成有关,但其信号机制及功能尚不完全明了. 目的 探讨利用小发卡环核糖核酸( shRNA)使人RPE细胞缺氧诱导因子1α(HIF-1α)基因沉默后,IGF-1对VEGF表达的影响. 方法 收集健康男性供体眼球4只,分离、收集、培养RPE细胞,用SABC法行抗人角蛋白免疫细胞化学染色进行鉴定.用体外转录法合成针对HIF-1α mRNA序列靶点的shRNA,对3～5代RPE细胞的HIF-1α进行干扰后再经50 μg/L IGF-1处理24 h,采用逆转录聚合酶链反应(RT-PCR)法检测人RPE细胞中HIF-1α及VEGF mRNA的表达,采用Western blot法检测人RPE细胞中HIF-1α及VEGF蛋白的水平.结果 分离培养的细胞呈扁平不规则多角形,97％的细胞对人角蛋白呈阳性反应.50 μg/L IGF-1作用后,人RPE细胞HIF-1α mRNA表达量(1.49±0.18)与0 μg/L IGF-1组(1.46±0.17)比较差异无统计学意义(t=0.335,P=0.743),而HIF-1α蛋白表达量(1049.86±172.54 vs 0.00±0.00)、VEGF mRNA(0.95±0.15 vs 0.35±0.07)及VEGF蛋白(391.98±56.77 vs 214.36±37.15)表达量均明显增高,差异均有统计学意义(t=16.098、9.935、6.928,P＜0.05).shRNA干扰HIF-1α mRNA表达后,RNAi转染组HIF-1α、VEGF mRNA及其蛋白水平较RNAi空白对照组及RNAi-C转染组明显下降,3个组间各指标的总体比较差异均有统计学意义(F=68.679、89.904、21.770、6.205,P＜0.05). 结论 IGF-1可通过促进人RPE细胞中HIF-1α蛋白的累积诱导VEGF的表达,是导致PVD重要的细胞因子之一.     

Inhibition of laser-induced choroidal neovascularization by atorvastatin by downregulation of monocyte chemotactic protein-1 synthesis in mice

PURPOSE: To determine the effect of atorvastatin, an HMG CoA reductase inhibitor, on experimental choroidal neovascularization (CNV) induced by laser photocoagulation in mice. METHODS: CNV was induced by laser photocoagulation in normal wild-type mice. The mice received either oral atorvastatin 10 (AS10 group) or 20 (AS20 group) mg/kg per day 3 days before and after laser application; 1 (AS1) and 2 (AS2) mg/kg per day were included in the measurement of the parameters of CNV volume and the expression of chemoattractant CC chemokine ligand 2/monocyte chemoattractant protein-1 (CCL2/MCP-1) and intracellular adhesion molecule (ICAM)-1. CNV responses were compared based on volume measurements 2 weeks after laser photocoagulation. Expression of vascular endothelial growth factor (VEGF), CCL2/MCP-1, and ICAM-1 in the RPE and choroid was quantified by ELISA 2 or 3 days after photocoagulation. Macrophage infiltration of the choroid was determined by flow cytometry. RESULTS: The mean CNV volume was significantly smaller in the AS1 (44.16 +/- 4.67 x 10(4) microm(3)), AS2 (36.49 +/- 4.64 x 10(4) microm(3)), AS10 (25.75 +/- 2.41 x 10(4) microm(3)), and AS20 (33.24 +/- 8.42 x 10(4) microm(3)) groups compared with control mice (64.21 +/- 2.27 x 10(4) microm(3); P = 0.0004, P < 0.0001, P < 0.0001, P < 0.0001, respectively). The mean VEGF and CCL2/MCP-1 protein levels decreased significantly (P = 0.001, P = 0.02, respectively) in the treated group compared with the control group. ICAM-1 expression did not differ significantly between the treated and control groups. The number of choroid-infiltrating macrophages decreased markedly in the treated group. CONCLUSIONS: Atorvastatin effectively inhibited laser-induced CNV in mice and was associated with downregulation of CCL2/MCP-1 and VEGF and reduced macrophage infiltration into the RPE/choroid.     

大鼠视网膜胚胎发育过程中低氧诱导因子-1α及血管内皮生长因子的表达

背景研究发现低氧诱导因子-1（HIF-1）与机体缺氧的生理反应有关,并在胚胎发育过程中发挥作用。此外人视网膜胚胎发育过程中血管内皮生长因子（VEGF）呈高表达。但二者在胚胎期缺氧环境中的作用及相互关系仍有待研究。目的观察大鼠视网膜胚胎发育过程中HIF-1α和VEGF的表达变化,探讨HIF-1α和VEGF在视网膜胚胎发育过程中的作用和相互关系。方法30只清洁级SD孕鼠剖腹取出胚胎10、12、14、16、20d鼠,每组取5只孕鼠,每只孕鼠随机选取2只胎鼠进行观察。摘除胎鼠一侧眼球,分离视网膜并制备切片,用免疫组织化学法检测视网膜组织中HIF-1α、VEGF蛋白的阳性表达,采用逆转录聚合酶链反应（RT—PCR）法分别检测不同胎龄大鼠及成年大鼠视网膜组织中HIF-1α mRNA及VEGF mRNA的表达。结果免疫组织化学染色表明,在大鼠视网膜胚胎早期（10～12d）即可检测到HIF-1α和VEGF蛋白均呈高表达,二者的表达强度均随胎龄的增加而减弱（F=56．70,P〈0．01;F=60．78,P〈0．01）,各胎龄组视网膜中HIF-1α和VEGF蛋白的表达量均明显高于成年鼠,差异均有统计学意义（P〈0．01）,随胎龄的增加HIF-1α蛋白在视网膜中的表达变化与VEGF蛋白表达呈正相关（r=0．96,P=0．00）。RT—PCR检测结果表明,大鼠胚胎早期（10～12d）即可检测到HIF-1α mRNA和VEGF mRNA在视网膜中均呈高表达,二者的表达均随胎龄的增加而减弱（F：68．84,P〈0．01;F=96．49,P〈0．01）,各胎龄组视网膜中HIF-1α和VEGF蛋白的表达量均明显高于成年鼠,差异均有统计学意义（P〈0．01）。结论大鼠视网膜胚胎发育过程中HIF-1α及VEGF的表达随着胎龄的增加均呈先高后低的动态变化趋势,提示HIF-1α／VEGF通路参与大鼠视网膜的胚胎发育过程。     

塞来昔布对实验性脉络膜新生血管的抑制作用

目的　观察选择性环氧化酶-2 (COX-2)抑制剂塞来昔布（celecoxib）对实验性脉络膜新生血管（CNV）的抑制作用。方法　鼠龄8~10周的健康雄性棕色挪威（BN）大鼠30只,随机分为空白对照组、实验对照组和塞来昔布治疗组,每组10只。塞来昔布治疗组采用灌胃法给药,剂量50 mg/kg,2次/d。给药后7 d,采用氪激光建立大鼠CNV模型,分别于激光光凝后3、7、14、21、30 d对实验对照组和塞来昔布治疗组所有大鼠行荧光素眼底血管造影(FFA)检查。激光光凝后21 d,每组随机处死5只大鼠,摘除眼球,制作空白对照组球后段组织切片、实验对照组和治疗组CNV膜切片。常规苏木精-伊红（HE）染色,计算实验对照组和塞来昔布治疗组的CNV膜相对厚度;采用免疫组织化学方法检测COX-2、血管内皮生长因子（VEGF）及基质金属蛋白酶-2（MMP-2)的表达。结果　激光光凝后21 d,塞来昔布治疗组 CNV发生率明显低于实验对照组（χ2=7.106 8,P=0.007 7）,CNV相对厚度较实验对照组明显减少（t=16.760 0,P=0.000 0）,COX-2、VEGF和MMP-2在CNV膜上的阳性表达均明显低于实验对照组（t=5.710 0,5.840 0,8.020 0;P=0.000 0）;空白对照组大鼠COX-2、VEGF和MMP-2在视网膜和脉络膜中的表达非常弱。结论　预防性服用塞来昔布能抑制激光诱导CNV的发生;通过抑制COX-2可减少CNV中VEGF和MMP-2的表达。