凉血化瘀方治疗湿性老年性黄斑变性回顾性临床研究及作用机制探讨
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摘要
[目的]1.总结分析凉血化瘀方加减对渗出性年龄相关性黄斑变性(age related macular degeneration, AMD)的治疗作用及远期效果。
2.探讨凉血化瘀方成药(明睛颗粒)对实验性脉络膜新生血管(choroidal neovascularization, CNV)的疗效观察及作用机制。
[方法]1.回顾性总结分析2005年1月—2010年4月到唐由之研究员门诊就诊且至少一只眼诊断为渗出性AMD的单纯服用凉血化瘀加减方全部患者,设定纳入、排除标准,对初诊及治疗后1个月、3个月、6个月、12个月患者视力、眼底情况、OCT分别评价分析。2.采用视网膜下注射Matrigel的方法建立实验性CNV模型,通过眼底彩照、FFA、脉络膜铺片、组织病理改变分别进行评价分析,确定Matrigel最佳浓度及操作方法。3.将40只青紫蓝兔随机分为空白对照组、模型组、明睛颗粒高、中、低剂量组,视网膜下注射Matrigel后连续观察28天,通过眼底彩照、FrA、组织切片对治疗效果进行评价；通过免疫组织化学检测CD14和CD206阳性细胞在玻璃体、视网膜及脉络膜的表达情况。4.以SPF级70只C57BL/6小鼠作为研究对象,以激光诱导CNV形成作为模型建立方法,采用冰冻组织切片及免疫荧光双标技术观察新生血管在激光斑处的增生情况、VEGF的表达以及巨噬细胞的分布及表型分析,观察明睛颗粒对上述指标的影响作用。
[结果]1.接受凉血化瘀方加减治疗渗出性AMD的患者1个月后视力即有显著性提高(p<0.05),连续治疗3个月视力提高大于两行者占16.13%,眼底出血及渗出的情况在84.34%患者中有好转乃至痊愈,平均视网膜厚度在3个月时也有明显减少(p<0.05)。2.从FFA来看,Matrigel40μl和20μl组从7天起出现荧光渗漏,14、28天逐渐增强；10μl组14和28天出现荧光渗漏,5ul组一直未见明显荧光素渗漏。从眼底情况来看,7天时大部分注射部位的视网膜已贴附回去,仅个别眼出现局限性视网膜脱离,Matrigel40μl组个别眼视网膜增殖明显,造成牵拉性视网膜脱离。因白内障或玻璃体浑浊无法进行眼底彩照和造影多出现在Matrigel40μl组。从组织病理学观察,小血管和/或幼稚纤维组织增生多见于Matrigel40μl和20μl组。但Matrigel40μl组视网膜损伤明显。3.从FFA来看,明睛颗粒大、中、小三个剂量均能有效减少病变部位荧光渗漏的面积(p<0.05)。从组织病理学评价来看,明睛颗粒对视网膜下注射Matrigel诱发受试动物眼底病变有一定的治疗作用,中剂量组效果最明显,低剂量组和高剂量组均次之。从免疫组化标记单核细胞(CD14+)和Ⅱ型巨噬细胞(CD206+)结果来看,模型组CD14+细胞明显增多(p<0.05),而CD206+细胞较少；与模型组比较,明睛颗粒治疗组的CD14+细胞没有明显减少,但CD206+细胞显著性增多(p<0.05)。4.在激光诱导实验性CNV模型中,造模后7天可见CD105+内皮细胞(新生血管)少量表达,14、28天逐渐增多。VEGF在新生血管团附近7天时有一定表达,11天时最明显，28天时有所减少。F4/80+细胞(成熟巨噬细胞标志物)在新生血管团附近随时问延长逐渐增多,以CD32+细胞(Ⅰ型巨噬细胞)为主,28天时可见少量CD206+细胞。明睛颗粒干预治疗后可明显控制新生血管的增生,减少VEGF表达,病变处以CD206+细胞为主。
[结论]1.凉血化瘀方通过快速有效地减少视网膜出血、渗出及水肿从而在一定程度上改善视力,观察12个月视力保持稳定。2.Matrigel视网膜下注射可成功诱导CNV形成,20μl为最佳剂量。3.明睛颗粒大、中、小三个剂量对CNV的形成和生长均有一定抑制作用,中剂量效果最好。4Matrigel视网膜下注射及激光诱导CNV形成两种动物模型均可见巨噬细胞参与,以Ⅰ型巨噬细胞为主；明睛颗粒可减少病变部位VEGF表达,诱导巨噬细胞向Ⅱ型转化。
[Objective]1. To sum up and analyze the long-term therapeutic effect of modified Liangxuehuayu formula on the treatment of exudative age related macular degeneration.2. To study the effect and the mechanism of Mingjing granule on the experimental CNV.
[Methods]1. Patients from Jan.2005to April2010treated by Dr. Tang youzhi with Liangxuehuayu formula for exudative age-related macular degeneration were reviewed and assessed. Bringing in and ruling out criterias were set up. Visual acuity, retinal manifestation and OCT were measured at first visit,1month after treatment (AT),3months AT,6months AT and12months AT.2. The method of subretinal injection of Matrigel was used to build up the model of experimental CNV. Fundus examination, FFA, choroidal flat mount and pathological changes were measured to choose the best dose and injecting technique of Matrigel.3.40grey rabbits were randomly divided into control group, model group, high dose of Mingjingkeli group, middle dose of Mingjingkeli group and small dose of Mingjingkeli group. The latter4groups were subretinal injected with Matrigel.28days later, fundus examination, FFA, tissue slides were measured; CD14and CD206possitive cells were measured by immunohistochemistry.4.70C57BL/6mice were used to build up the laser induced CNV model, frost tissue slides and immunofluorescence double labers techniques were used to measure the CNV growth、VEGF expression、macrophages and its phenotype around laser spots, the effect of Mingjingkili on the above aspects was also valued.
[Results]1. Visual acuity of patients who received the treatment of Liangxuehuayu formula was improved after1month therapy(p<0.05), the rate of visual acuity increased at least two lines after3months treatment was16.13%. Retinal hemorrhage and exudation was alleviated or cured among84.34%patients, average retinalthickness was also extenuated after3months treatment (p<0.05).2. In the FFA examination, the fluorescence leakage was happened in Matrigel40μl and20μl groups at7d, which was enhanced at14d and28d; the fluorescence leakage was happened in Matrigel10μl group at14d and28d, which was never found in Matrigel5μl group.In the fundus examination, most of retina were attached back at7d, a few of those were detached. A few of retina had proliferative detachment in Matrigel40μl group. Cataract and vitreous muddy were found in Matrigel40μl group. In the tissue pathological examination, small vessels and/or immature fibrous tissues were found in Matrigel40μl and20μl groups. The retinal injure was most severely in Matrigel40μl group.3. In the FFA examination, all of the large, middle and small doses of Mingjingkeli could decrease the area of fluorescence leakage (p<0.05). In the tissue pathological examination, Mingjingkeli had the protective effect on the retina which was injected with Matrigel, the middle dose is the best, the large and small doses were the second. Based on the immunohistochemistry, there had many CD14+cells(monocyte)(p<0.05), less CD206+cells(type Ⅱ macrophage) in model group, while there had the same CD14+cells and much more CD206+cells in Mingjingkeli group(p<0.05).4. In the laser induced experimental CNV model, CD105+endothelial cells could be seen in7d, increased in14d and28d. VEGF was seen in neovascular masses in7d, was most obvious in14d and decreased in28d. F4/80+cells (mature macrophages) in neovascular masses were increased with time, most of those were CD32+cells (type Ⅰ macrophages), there were some CD206+cells in28d in model group. Mingjingkeli could decrease the growth of neovascularization, decrease the expression of VEGF, most of macrophages were CD206+cells.
[Conclusion]1. Liangxuehuayu formula could improve the visual acuity by diminishing the retinal hemorrhage, exudation and edema, the visual acuity could be kept stable within12months.2. The method of subretinal injection of Matrigel could create CNV successfully,20μl is the best dose.3. All three doses of Mingjingkeli could inhibit CNV growth, the middle dose is the best one.4. Macrophages could be seen in the formation of CNV in both models of subretinal injection of Matrigel and laser induced CNV, most of which are type Ⅰ macrophages; Mingjingkeli could decrease the expression of VEGF, derivate the production of type Ⅱ macrophages.
2.探讨凉血化瘀方成药(明睛颗粒)对实验性脉络膜新生血管(choroidal neovascularization, CNV)的疗效观察及作用机制。
[方法]1.回顾性总结分析2005年1月—2010年4月到唐由之研究员门诊就诊且至少一只眼诊断为渗出性AMD的单纯服用凉血化瘀加减方全部患者,设定纳入、排除标准,对初诊及治疗后1个月、3个月、6个月、12个月患者视力、眼底情况、OCT分别评价分析。2.采用视网膜下注射Matrigel的方法建立实验性CNV模型,通过眼底彩照、FFA、脉络膜铺片、组织病理改变分别进行评价分析,确定Matrigel最佳浓度及操作方法。3.将40只青紫蓝兔随机分为空白对照组、模型组、明睛颗粒高、中、低剂量组,视网膜下注射Matrigel后连续观察28天,通过眼底彩照、FrA、组织切片对治疗效果进行评价；通过免疫组织化学检测CD14和CD206阳性细胞在玻璃体、视网膜及脉络膜的表达情况。4.以SPF级70只C57BL/6小鼠作为研究对象,以激光诱导CNV形成作为模型建立方法,采用冰冻组织切片及免疫荧光双标技术观察新生血管在激光斑处的增生情况、VEGF的表达以及巨噬细胞的分布及表型分析,观察明睛颗粒对上述指标的影响作用。
[结果]1.接受凉血化瘀方加减治疗渗出性AMD的患者1个月后视力即有显著性提高(p<0.05),连续治疗3个月视力提高大于两行者占16.13%,眼底出血及渗出的情况在84.34%患者中有好转乃至痊愈,平均视网膜厚度在3个月时也有明显减少(p<0.05)。2.从FFA来看,Matrigel40μl和20μl组从7天起出现荧光渗漏,14、28天逐渐增强；10μl组14和28天出现荧光渗漏,5ul组一直未见明显荧光素渗漏。从眼底情况来看,7天时大部分注射部位的视网膜已贴附回去,仅个别眼出现局限性视网膜脱离,Matrigel40μl组个别眼视网膜增殖明显,造成牵拉性视网膜脱离。因白内障或玻璃体浑浊无法进行眼底彩照和造影多出现在Matrigel40μl组。从组织病理学观察,小血管和/或幼稚纤维组织增生多见于Matrigel40μl和20μl组。但Matrigel40μl组视网膜损伤明显。3.从FFA来看,明睛颗粒大、中、小三个剂量均能有效减少病变部位荧光渗漏的面积(p<0.05)。从组织病理学评价来看,明睛颗粒对视网膜下注射Matrigel诱发受试动物眼底病变有一定的治疗作用,中剂量组效果最明显,低剂量组和高剂量组均次之。从免疫组化标记单核细胞(CD14+)和Ⅱ型巨噬细胞(CD206+)结果来看,模型组CD14+细胞明显增多(p<0.05),而CD206+细胞较少；与模型组比较,明睛颗粒治疗组的CD14+细胞没有明显减少,但CD206+细胞显著性增多(p<0.05)。4.在激光诱导实验性CNV模型中,造模后7天可见CD105+内皮细胞(新生血管)少量表达,14、28天逐渐增多。VEGF在新生血管团附近7天时有一定表达,11天时最明显，28天时有所减少。F4/80+细胞(成熟巨噬细胞标志物)在新生血管团附近随时问延长逐渐增多,以CD32+细胞(Ⅰ型巨噬细胞)为主,28天时可见少量CD206+细胞。明睛颗粒干预治疗后可明显控制新生血管的增生,减少VEGF表达,病变处以CD206+细胞为主。
[结论]1.凉血化瘀方通过快速有效地减少视网膜出血、渗出及水肿从而在一定程度上改善视力,观察12个月视力保持稳定。2.Matrigel视网膜下注射可成功诱导CNV形成,20μl为最佳剂量。3.明睛颗粒大、中、小三个剂量对CNV的形成和生长均有一定抑制作用,中剂量效果最好。4Matrigel视网膜下注射及激光诱导CNV形成两种动物模型均可见巨噬细胞参与,以Ⅰ型巨噬细胞为主；明睛颗粒可减少病变部位VEGF表达,诱导巨噬细胞向Ⅱ型转化。
[Objective]1. To sum up and analyze the long-term therapeutic effect of modified Liangxuehuayu formula on the treatment of exudative age related macular degeneration.2. To study the effect and the mechanism of Mingjing granule on the experimental CNV.
[Methods]1. Patients from Jan.2005to April2010treated by Dr. Tang youzhi with Liangxuehuayu formula for exudative age-related macular degeneration were reviewed and assessed. Bringing in and ruling out criterias were set up. Visual acuity, retinal manifestation and OCT were measured at first visit,1month after treatment (AT),3months AT,6months AT and12months AT.2. The method of subretinal injection of Matrigel was used to build up the model of experimental CNV. Fundus examination, FFA, choroidal flat mount and pathological changes were measured to choose the best dose and injecting technique of Matrigel.3.40grey rabbits were randomly divided into control group, model group, high dose of Mingjingkeli group, middle dose of Mingjingkeli group and small dose of Mingjingkeli group. The latter4groups were subretinal injected with Matrigel.28days later, fundus examination, FFA, tissue slides were measured; CD14and CD206possitive cells were measured by immunohistochemistry.4.70C57BL/6mice were used to build up the laser induced CNV model, frost tissue slides and immunofluorescence double labers techniques were used to measure the CNV growth、VEGF expression、macrophages and its phenotype around laser spots, the effect of Mingjingkili on the above aspects was also valued.
[Results]1. Visual acuity of patients who received the treatment of Liangxuehuayu formula was improved after1month therapy(p<0.05), the rate of visual acuity increased at least two lines after3months treatment was16.13%. Retinal hemorrhage and exudation was alleviated or cured among84.34%patients, average retinalthickness was also extenuated after3months treatment (p<0.05).2. In the FFA examination, the fluorescence leakage was happened in Matrigel40μl and20μl groups at7d, which was enhanced at14d and28d; the fluorescence leakage was happened in Matrigel10μl group at14d and28d, which was never found in Matrigel5μl group.In the fundus examination, most of retina were attached back at7d, a few of those were detached. A few of retina had proliferative detachment in Matrigel40μl group. Cataract and vitreous muddy were found in Matrigel40μl group. In the tissue pathological examination, small vessels and/or immature fibrous tissues were found in Matrigel40μl and20μl groups. The retinal injure was most severely in Matrigel40μl group.3. In the FFA examination, all of the large, middle and small doses of Mingjingkeli could decrease the area of fluorescence leakage (p<0.05). In the tissue pathological examination, Mingjingkeli had the protective effect on the retina which was injected with Matrigel, the middle dose is the best, the large and small doses were the second. Based on the immunohistochemistry, there had many CD14+cells(monocyte)(p<0.05), less CD206+cells(type Ⅱ macrophage) in model group, while there had the same CD14+cells and much more CD206+cells in Mingjingkeli group(p<0.05).4. In the laser induced experimental CNV model, CD105+endothelial cells could be seen in7d, increased in14d and28d. VEGF was seen in neovascular masses in7d, was most obvious in14d and decreased in28d. F4/80+cells (mature macrophages) in neovascular masses were increased with time, most of those were CD32+cells (type Ⅰ macrophages), there were some CD206+cells in28d in model group. Mingjingkeli could decrease the growth of neovascularization, decrease the expression of VEGF, most of macrophages were CD206+cells.
[Conclusion]1. Liangxuehuayu formula could improve the visual acuity by diminishing the retinal hemorrhage, exudation and edema, the visual acuity could be kept stable within12months.2. The method of subretinal injection of Matrigel could create CNV successfully,20μl is the best dose.3. All three doses of Mingjingkeli could inhibit CNV growth, the middle dose is the best one.4. Macrophages could be seen in the formation of CNV in both models of subretinal injection of Matrigel and laser induced CNV, most of which are type Ⅰ macrophages; Mingjingkeli could decrease the expression of VEGF, derivate the production of type Ⅱ macrophages.
引文
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[27]汪振芳,万鹏霞,何丽文,袁钊辉,温容,大鼠视网膜下注射Matrigel诱导视网膜色素上皮易位和脉络膜新生血管形成.中华生物医学工程杂志,2007,13(6)：345-8
[28]Guanting Qiu, Jay M. Stewart, SriniVas Sadda, Ricardo Freda, Steven Lee, Dilek Guven, Eugene de Juan Jr., Signe E. Varner. A new model of experimental subretinal neovascularization in the rabbit. Experimental Eye Research 83 (2006) 141-52.
[29]Mantovani A,Sica A,Sozzani S,et al.The chemokine system in diverse forms of macrophage activation and polarization.Trends Immunol,2004,12(12):677-86
[1]Grossniklaus HE, Hutchinson AK, Capone Jr A, Woolfson J, Lambert HM.Clinicopathologic features of surgically excised choroidal neovascular membranes. Ophthalmology 1994; 101:1099-111.
[2]JM Skeie, RF Mullins. Macrophages in neovascular age-related macular degeneration:friends or foes? Eye (2009) 23,747-755
[3]Blumenkranz M, Russell S, Robey M, Kott-Blumenkranz R, Penneys N. Risk factors in age-related maculopathy complicated by choroidal neovascularization. Ophthalmology 1986; 93:552-558.
[4jCousins S, Espinosa-Heidmann D, Csaky K. Monocyte activation in patients with age-related macular degeneration:a biomarker of risk for choroidal neovascularization. Arch Ophthalmol 2004; 122:1013-1018.
[5]Seddon J, Gensler G, Milton R, Klein M, Rifai N. Association between C-reactive protein and age-related macular degeneration. JAMA 2004; 291:704-710.
[6]Seddon JM, George S, Rosner B, Rifai N. Progression of age-related macular degeneration:prospective assessment of C-reactive protein, interleukin 6, and other cardiovascular biomarkers. Arch Ophthalmol 2005; 123(6):774-782.
[7]Ranson NT, Danis RP, Ciulla TA, Pratt L. Intravitreal triamcinolone in subfoveal recurrence of choroidal neovascularisation after laser treatment in macular degeneration. Br J Ophthalmol 2002; 86(5):527-529.
[8]Tuo J, Smith BC, Bojanowski CM, Meleth AD, Gery I, Csaky KG et al. The involvement of sequence variation and expression of CX3CR1 in the pathogenesis of age-related macular degeneration. FASEB J 2004; 18(11):1297-1299.
[9]Goverdhan S, Mullins R, Howell W, Osmond C, Hodgkins P, Self J et al. Association of HLA class Ⅰ and class Ⅱ polymorphisms with age-related macular degeneration. Invest Ophthalmol Vis Sci 2005; 46(5):1726-1734.
[10]Gold B, Merriam JE, Zernant J, Hancox LS, Taiber AJ, Gehrs K et al. Variation in factor B (BF) and complement component 2 (C2) genes is associated with age-related macular egeneration. Nat Genet 2006; 38(4):458-462.
[11]Penfold P, Provis J, Billson F. Age-related macular degeneration: ultrastructural studies of the relationship of leukocytes to angiogenesis. Graefes Arch Clin Exp Ophthalmol 1987; 225:70-76.
[12]Lopez P, Grossniklaus H, Lambert H, Aaberg T, Capone A, Sternberg P et al. Pathologic features of surgically excised subretinal neovascular membranes in age-related macular degeneration. Am J Ophthalmol 1991; 112:647-656.
[13]Grossniklaus H, Green W, Submacular Surgery Trials Research Group. Histopathologic and ultrastructural findings of surgically excised choroidal neovascularization. Arch Ophthalmol 1998; 116: 745-749.
[14]Grossniklaus H, Cingle K, Yoon Y, Ketkar N, L'Hernault N, Brown S. Correlation of histologic 2-dimensional reconstruction and confocal scanning laser microscopic imaging of choroidal neovascularization in eyes with agerelated maculopathy. Arch Ophthalmol 2000; 118:625-629.
[15]Grossniklaus HE, Ling JX, Wallace TM, Dithmar S, Lawson DH, Cohen C et al. Macrophage and retinal pigment epithelium expression of angiogenic cytokines in choroidal neovascularization. Mol Vis 2002; 8:119-126.
[16]Radi Z, Kehrli M, Ackermann M. Cell adhesion molecules, leukocyte trafficking, and strategies to reduce leukocyte infiltration. J Vet Intern Med 2001; 15:516-529.
[17]Mullins R, Skeie J, Malone E, Kuehn M. Macular and peripheral distribution of ICAM-1 in the human choriocapillaris and retina. Mol Vis 2006; 12:224-235.
[18]Szekanecz Z, Koch A. Macrophages and their products in rheumatoid arthritis. Curr Opin Rheumatol 2006; 19:289-295.
[19]Otani A, Kinder K, Ewalt K, et al. Bone marrow2derived stem cells target retinal ast rocytes and can promote or inhibit retinal angiogenesis [J]. Nat Med,2002,8 (9):1004-1010.
[20]Espinosa2Heidmann DG, Caicedo A, Hernandez EP, et al Cousins1bone marrow2derived progenitor cells cont ribute to experimental choroidal neovascularization. Invest Ophthalmol Vis Sci, 2003,44(11):4914-4919
[21]Espinosa-Heidman D, Suner I, Hernandez E, Monroy D, Csaky K, Cousins S. Macrophage depletion diminishes lesion size and severity in experimental choroidal neovascularization. Invest Ophthalmol Vis Sci 2003; 44(8):3586-3592.
[22]Grossniklaus HE, Ling JX, Wallace TM, Dithmar S, Lawson DH, Cohen C et al. Macrophage and retinal pigment epithelium expression of angiogenic cytokines in choroidal neovascularization. Mol Vis 2002; 8:119-126.
[23]Tsutsumi C, Sonoda KH, Egashira K, Qiao H, Hisatomi T, Nakao S et al. The critical role of ocular-infiltrating macrophages in the development of choroidal neovascularization. J Leukoc Biol 2003; 74(1): 25-32.
[24]Sakurai E, Taguchi H, Anand A, Ambati B, Gragoudas E, Miller J et al. Targeted disruption of the CD18 or ICAM-1 gene inhibits choroidal neovascularization. Invest Ophthalmol Vis Sci 2003; 44: 2743-2749.
[25]Holt kamp GM, De Vos AF, Peek R, et al. Analysis of t he secretion pattern of monocyte chemotactic protein21 (MCP21) and transforming growth factor2beta 2 (TGF2β2) by human retinal pigment epithelial cells[J]. Clin Exp Immunol,1999,118 (1):35-40.
[26]Sakurai E, Anand A, Ambati BK, et al. Macrophage depletion inhibits experimental choroidal neovascularization [J]. Invest Ophthalmol Vis Sci,2003,44 (8):3578-3585.
[27]Ishibashi T, Hata Y, Yoshikawa H, et al. Expression of vascular endothelial growth factor in experimental choroidal neovascularization[J].Graefes Arch Clin Exp Opht halmol,1997,235 (3):159-167.
[28]Kiriakidis S, Andreakos E, Monaco C, et al. VEGF expression in human macrophages is NF2kB dependent:studies using adenoviruses expressing the endogenous NF2kB inhibitor IkBa and a kinasedefec2 tive form of t he IkB kinase 2[J]. J Cell Science,2003,116 (4):665-674.
[29]Apte RS, Richter J, Herndon J, Ferguson TA. Macrophages inhibit neovascularization in a murine model of age-related macular degeneration. PLoS Med 2006; 3(8):310.
[30]Mantovani A,Sica A,Sozzani S,et al.The chemokine system in diverse forms of macrophage activation and polarization.Trends Immunol,2004,12(12):677-86
[31]Verreck FAW,Boer TD,Langenberg DML,et al.Phenotypic and functional profiling of human proinflammatory type-1 and anti-inflammatory type-2 macrophages in response to microbial antigens and IFN-yand CD40L-mediated costimulation.J Leukoc Biol,2006,79(2): 285-93
[32]Mosser DM.The many faces of macrophage activation.J Leukoc Biol,2003,73(2):209-12
[33]Gordon S, Taylor P. Monocyte and macrophage heterogeneity. Nat Rev Immunol 2005; 5:953-964.
[1]Al-Akily SA, Bamashmus MA, Al-Mohammadi KA. Causes of blindness in people aged 50 years and over:community-based versus hospital-based study[J].East Mediterr Health J.2010,16(9):942-6.
[2]李学晶,杨薇,唐由之,李博.中医药治疗年龄相关性黄斑变性文献的方法学质量评价[J].中国中医眼科杂志,2009,19(2)：83-6.
[3]谢雁鸣,王永炎,朴海垠.构建中医软指标疗效评价模式的初步探索[J].中国中西医结合杂志,2007,27(6)：560-564.
[4]胡伟,徐蕾,钱先,中医药治疗干燥综合征随机对照试验的质量评价[J].中国循证医学杂志,2011,11(8)：978-82.
[2]Lopez P, Grossniklaus H, Lambert H, Aaberg T, Capone A, Sternberg P et al. Pathologic features of surgically excised subretinal neovascular membranes in age-related macular degeneration. Am J Ophthalmol,1991,112:647-56.
[3]Grossniklaus H, Green W, Submacular Surgery Trials Research Group. Histopathologic and ultrastructural findings of surgically excised choroidal neovascularization. Arch Ophthalmol,1998,116: 745-9.
[4]Grossniklaus H, Cingle K, Yoon Y, Ketkar N, L'Hernault N, Brown S. Correlation of histologic 2-dimensional reconstruction and confocal scanning laser microscopic imaging of choroidal neovascularization in eyes with agerelated maculopathy. Arch Ophthalmol,2000,118:625-9.
[5]Grossniklaus HE, Ling JX, Wallace TM, Dithmar S, Lawson DH, Cohen C et al. Macrophage and retinal pigment epithelium expression of angiogenic cytokines in choroidal neovascularization. Mol Vis,2002,8: 119-26.
[6]Mullins R, Skeie J, Malone E, Kuehn M. Macular and peripheral distribution of ICAM-1 in the human choriocapillaris and retina. Mol Vis,2006,12:224-35.
[7]Szekanecz Z, Koch A. Macrophages and their products in rheumatoid arthritis. Curr Opin Rheumatol,2006,19:289-95.
[8]Espinosa-Heidman D, Suner I, Hernandez E, Monroy D, Csaky K, Cousins S. Macrophage depletion diminishes lesion size and severity in experimental choroidal neovascularization. Invest Ophthalmol Vis Sci, 2003,44(8):3586-92.
[9]Grossniklaus HE, Ling JX, Wallace TM, Dithmar S, Lawson DH, Cohen C et al. Macrophage and retinal pigment epithelium expression of angiogenic cytokines in choroidal neovascularization. Mol Vis,2002,8: 119-26.
[10]Tsutsumi C, Sonoda KH, Egashira K, Qiao H, Hisatomi T, Nakao S et al. The critical role of ocular-infiltrating macrophages in the development of choroidal neovascularization. J Leukoc Biol,2003, 74(1):25-32.
[11]Sakurai E, Taguchi H, Anand A, Ambati B, Gragoudas E, Miller J et al. Targeted disruption of the CD18 or ICAM-1 gene inhibits choroidal neovascularization. Invest Ophthalmol Vis Sci,2003,44: 2743-9.
[12]Holt kamp GM, De Vos AF, Peek R, et al. Analysis of the secretion pattern of monocyte chemotactic protein21 (MCP21) and transforming growth factor2beta 2 (TGF2(32) by human retinal pigment epithelial cells. Clin Exp Immunol,1999,118 (1):35-40.
[13]Sakurai E, Anand A, Ambati BK, et al. Macrophage depletion inhibits experimental choroidal neovascularization. Invest Ophthalmol Vis Sci,2003,44 (8):3578-85.
[14]Apte RS, Richter J, Herndon J, Ferguson TA. Macrophages inhibit neovascularization in a murine model of age-related macular degeneration. PLoS Med,2006,3(8):310.
[15]王慧娟,唐由之.凉血化瘀方治疗湿性老年黄斑变性临床研究,中国中医眼科杂志,2008,18(6),322-4.
[16]李学晶,唐由之,王慧娟.不同浓度凉血化瘀中药对脉络膜新生血管形态学的影响。中国中西医结合杂志,2009,29(10)：896-900.
[17]李学晶,唐由之.凉血化瘀中药与曲安奈德对脉络膜新生血管动物模型中MMP-9及TIMP-2表达的影响,国际眼科杂志,2009,9(4)：656-9.
[18]唐由之,王慧娟,冯俊.凉血化瘀方对实验性脉络膜新生血管VEGF与PEDF表达的影响。眼科新进展,2009,29(12)：881-5.
[19]于静,冯俊,王津津,张励,唐由之.凉血化瘀方对蓝光诱导人视网膜色素上皮细胞凋亡的影响。中国中医眼科杂志,2008,18(2)：92-5.
[20]于静,唐由之,冯俊,陈燕云,张励.凉血化瘀方对人视网膜色素上皮细胞吞噬光感受器外节作用影响的研究。辽宁中医杂志,2009,36(12)：2038-41.
[21]张承芬,眼底病学,人民卫生出版社,1998：332
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[23]郑曰忠,时冀川.视力的检测和统计方法.中华眼科杂志,2002,38(2)：67-8
[24]丛珊,李凡.研究中不等距重复测量资料的分析及在SPSS16.0中的实现数理医药学杂志,2009,22(6)：649-52
[25]Rosenfeld PJ, Brown DM, Heier JS, et al. Ranibizumab for neovascular age-related macular degeneration. N Engl J Med.2006, 355:1419-31.
[26]Brown DM, Kaiser PK, Michels M, et al. Ranibizumab versus verteporfin for neovascular age-related macular degeneration. N Engl J Med.2006,355:1432-44.
[27]汪振芳,万鹏霞,何丽文,袁钊辉,温容,大鼠视网膜下注射Matrigel诱导视网膜色素上皮易位和脉络膜新生血管形成.中华生物医学工程杂志,2007,13(6)：345-8
[28]Guanting Qiu, Jay M. Stewart, SriniVas Sadda, Ricardo Freda, Steven Lee, Dilek Guven, Eugene de Juan Jr., Signe E. Varner. A new model of experimental subretinal neovascularization in the rabbit. Experimental Eye Research 83 (2006) 141-52.
[29]Mantovani A,Sica A,Sozzani S,et al.The chemokine system in diverse forms of macrophage activation and polarization.Trends Immunol,2004,12(12):677-86
[1]Grossniklaus HE, Hutchinson AK, Capone Jr A, Woolfson J, Lambert HM.Clinicopathologic features of surgically excised choroidal neovascular membranes. Ophthalmology 1994; 101:1099-111.
[2]JM Skeie, RF Mullins. Macrophages in neovascular age-related macular degeneration:friends or foes? Eye (2009) 23,747-755
[3]Blumenkranz M, Russell S, Robey M, Kott-Blumenkranz R, Penneys N. Risk factors in age-related maculopathy complicated by choroidal neovascularization. Ophthalmology 1986; 93:552-558.
[4jCousins S, Espinosa-Heidmann D, Csaky K. Monocyte activation in patients with age-related macular degeneration:a biomarker of risk for choroidal neovascularization. Arch Ophthalmol 2004; 122:1013-1018.
[5]Seddon J, Gensler G, Milton R, Klein M, Rifai N. Association between C-reactive protein and age-related macular degeneration. JAMA 2004; 291:704-710.
[6]Seddon JM, George S, Rosner B, Rifai N. Progression of age-related macular degeneration:prospective assessment of C-reactive protein, interleukin 6, and other cardiovascular biomarkers. Arch Ophthalmol 2005; 123(6):774-782.
[7]Ranson NT, Danis RP, Ciulla TA, Pratt L. Intravitreal triamcinolone in subfoveal recurrence of choroidal neovascularisation after laser treatment in macular degeneration. Br J Ophthalmol 2002; 86(5):527-529.
[8]Tuo J, Smith BC, Bojanowski CM, Meleth AD, Gery I, Csaky KG et al. The involvement of sequence variation and expression of CX3CR1 in the pathogenesis of age-related macular degeneration. FASEB J 2004; 18(11):1297-1299.
[9]Goverdhan S, Mullins R, Howell W, Osmond C, Hodgkins P, Self J et al. Association of HLA class Ⅰ and class Ⅱ polymorphisms with age-related macular degeneration. Invest Ophthalmol Vis Sci 2005; 46(5):1726-1734.
[10]Gold B, Merriam JE, Zernant J, Hancox LS, Taiber AJ, Gehrs K et al. Variation in factor B (BF) and complement component 2 (C2) genes is associated with age-related macular egeneration. Nat Genet 2006; 38(4):458-462.
[11]Penfold P, Provis J, Billson F. Age-related macular degeneration: ultrastructural studies of the relationship of leukocytes to angiogenesis. Graefes Arch Clin Exp Ophthalmol 1987; 225:70-76.
[12]Lopez P, Grossniklaus H, Lambert H, Aaberg T, Capone A, Sternberg P et al. Pathologic features of surgically excised subretinal neovascular membranes in age-related macular degeneration. Am J Ophthalmol 1991; 112:647-656.
[13]Grossniklaus H, Green W, Submacular Surgery Trials Research Group. Histopathologic and ultrastructural findings of surgically excised choroidal neovascularization. Arch Ophthalmol 1998; 116: 745-749.
[14]Grossniklaus H, Cingle K, Yoon Y, Ketkar N, L'Hernault N, Brown S. Correlation of histologic 2-dimensional reconstruction and confocal scanning laser microscopic imaging of choroidal neovascularization in eyes with agerelated maculopathy. Arch Ophthalmol 2000; 118:625-629.
[15]Grossniklaus HE, Ling JX, Wallace TM, Dithmar S, Lawson DH, Cohen C et al. Macrophage and retinal pigment epithelium expression of angiogenic cytokines in choroidal neovascularization. Mol Vis 2002; 8:119-126.
[16]Radi Z, Kehrli M, Ackermann M. Cell adhesion molecules, leukocyte trafficking, and strategies to reduce leukocyte infiltration. J Vet Intern Med 2001; 15:516-529.
[17]Mullins R, Skeie J, Malone E, Kuehn M. Macular and peripheral distribution of ICAM-1 in the human choriocapillaris and retina. Mol Vis 2006; 12:224-235.
[18]Szekanecz Z, Koch A. Macrophages and their products in rheumatoid arthritis. Curr Opin Rheumatol 2006; 19:289-295.
[19]Otani A, Kinder K, Ewalt K, et al. Bone marrow2derived stem cells target retinal ast rocytes and can promote or inhibit retinal angiogenesis [J]. Nat Med,2002,8 (9):1004-1010.
[20]Espinosa2Heidmann DG, Caicedo A, Hernandez EP, et al Cousins1bone marrow2derived progenitor cells cont ribute to experimental choroidal neovascularization. Invest Ophthalmol Vis Sci, 2003,44(11):4914-4919
[21]Espinosa-Heidman D, Suner I, Hernandez E, Monroy D, Csaky K, Cousins S. Macrophage depletion diminishes lesion size and severity in experimental choroidal neovascularization. Invest Ophthalmol Vis Sci 2003; 44(8):3586-3592.
[22]Grossniklaus HE, Ling JX, Wallace TM, Dithmar S, Lawson DH, Cohen C et al. Macrophage and retinal pigment epithelium expression of angiogenic cytokines in choroidal neovascularization. Mol Vis 2002; 8:119-126.
[23]Tsutsumi C, Sonoda KH, Egashira K, Qiao H, Hisatomi T, Nakao S et al. The critical role of ocular-infiltrating macrophages in the development of choroidal neovascularization. J Leukoc Biol 2003; 74(1): 25-32.
[24]Sakurai E, Taguchi H, Anand A, Ambati B, Gragoudas E, Miller J et al. Targeted disruption of the CD18 or ICAM-1 gene inhibits choroidal neovascularization. Invest Ophthalmol Vis Sci 2003; 44: 2743-2749.
[25]Holt kamp GM, De Vos AF, Peek R, et al. Analysis of t he secretion pattern of monocyte chemotactic protein21 (MCP21) and transforming growth factor2beta 2 (TGF2β2) by human retinal pigment epithelial cells[J]. Clin Exp Immunol,1999,118 (1):35-40.
[26]Sakurai E, Anand A, Ambati BK, et al. Macrophage depletion inhibits experimental choroidal neovascularization [J]. Invest Ophthalmol Vis Sci,2003,44 (8):3578-3585.
[27]Ishibashi T, Hata Y, Yoshikawa H, et al. Expression of vascular endothelial growth factor in experimental choroidal neovascularization[J].Graefes Arch Clin Exp Opht halmol,1997,235 (3):159-167.
[28]Kiriakidis S, Andreakos E, Monaco C, et al. VEGF expression in human macrophages is NF2kB dependent:studies using adenoviruses expressing the endogenous NF2kB inhibitor IkBa and a kinasedefec2 tive form of t he IkB kinase 2[J]. J Cell Science,2003,116 (4):665-674.
[29]Apte RS, Richter J, Herndon J, Ferguson TA. Macrophages inhibit neovascularization in a murine model of age-related macular degeneration. PLoS Med 2006; 3(8):310.
[30]Mantovani A,Sica A,Sozzani S,et al.The chemokine system in diverse forms of macrophage activation and polarization.Trends Immunol,2004,12(12):677-86
[31]Verreck FAW,Boer TD,Langenberg DML,et al.Phenotypic and functional profiling of human proinflammatory type-1 and anti-inflammatory type-2 macrophages in response to microbial antigens and IFN-yand CD40L-mediated costimulation.J Leukoc Biol,2006,79(2): 285-93
[32]Mosser DM.The many faces of macrophage activation.J Leukoc Biol,2003,73(2):209-12
[33]Gordon S, Taylor P. Monocyte and macrophage heterogeneity. Nat Rev Immunol 2005; 5:953-964.
[1]Al-Akily SA, Bamashmus MA, Al-Mohammadi KA. Causes of blindness in people aged 50 years and over:community-based versus hospital-based study[J].East Mediterr Health J.2010,16(9):942-6.
[2]李学晶,杨薇,唐由之,李博.中医药治疗年龄相关性黄斑变性文献的方法学质量评价[J].中国中医眼科杂志,2009,19(2)：83-6.
[3]谢雁鸣,王永炎,朴海垠.构建中医软指标疗效评价模式的初步探索[J].中国中西医结合杂志,2007,27(6)：560-564.
[4]胡伟,徐蕾,钱先,中医药治疗干燥综合征随机对照试验的质量评价[J].中国循证医学杂志,2011,11(8)：978-82.