凉血化瘀方对湿性年龄相关性黄斑变性的治疗观察及实验研究
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摘要
目的:
1.评价激光诱导棕色挪威(brown norway,BN)大鼠脉络膜新生血管(choroidalneovascularization,CNV)模型的可行性,并从荧光素眼底血管造影(fundus fluoresceinangiography,FFA)、脉络膜铺片血管定量分析、病理、血管内皮细胞免染色疫组化法进行多方面比较。
2.观察评价实验性脉络膜新生血管中血管内皮生长因子(vascular endothelialgrowth factor,VEGF)与色素上皮衍生因子(pigment epithelium-derived factor,PEDF)的表达水平及相互关系。
3.观察凉血化瘀方对激光诱导的BN大鼠CNV形成的抑制作用,从FFA检查、脉络膜铺片血管定量分析、病理、血管内皮细胞染色免疫组化法、VEGF与PEDF蛋白表达水平方面进行多方位评价。
4.观察凉血化瘀方治疗湿性年龄相关性黄斑变性(age-related macular degeneration,AMD)患者的临床疗效,评价指标有:视力、FFA检查、眼底出血面积、渗出面积、累积损害面积、黄斑视网膜厚度、黄斑区总容积。
方法:
1.对4组24只BN大鼠单眼进行氪激光视网膜光凝,围绕视盘等距光凝20个激光斑,诱导CNV形成。激光功率360mW,光斑直径50μm,曝光时间0.05s。分别在光凝后1w、2w、3w、4w进行眼底彩照和FFA检查。在上述时间点各取2只大鼠进行脉络膜铺片定量分析血管面积;余4只处死,取眼球做石蜡切片,观察激光斑病理改变,FⅧR:Ag(FactorⅧ-Related Antigen,Ⅷ因子相关抗原)免疫组化法对新生血管进行半定量分析。
2.通过石蜡切片观察CNV发展的4w时间内,不同时间点CNV中细胞因子VEGF和PEDF的蛋白表达。
3.对3组48只BN大鼠行氪激光光凝,建立CNV动物模型,其中1组作为空白组,1组造模后玻璃体腔注射曲安奈德做为对照组,1组造模后予以凉血化瘀方灌胃作为中药组。分别在光凝后1w、2w、3w、4w行FFA检查。在上述各时间点处死各组动物4只,其中1只行异硫氰酸荧光素葡聚糖(Fluorescein isothiocyanate dextran,FITC-D)灌注、脉络膜铺片定量新生血管面积,余3只摘出眼球,制作石蜡切片,观察不同时间点CNV的中FⅧR:Ag、VEGF、PEDF表达的变化,并进行半定量检测。
4.观察临床确诊为CNV型AMD的患者90例患者109眼,予以口服凉血化瘀中药治疗。连续观察6个月,总结视力,眼底出血面积、渗出面积、累积损害面积、OCT检查结果。分析比较治疗前后视力、眼底出血、渗出、累积损害面积、黄斑视网膜厚度和黄斑区总容积变化,并观察用药过程中不良反应。
结果:
1.在4w时间内,FFA检查、脉络膜铺片血管面积测量、病理切片、FⅧR:Ag因子免疫组化结果显示,实验性CNV在1w～3w期间呈持续性增殖(P＜0.05),到4w时稳定。
2.在实验性CNV形成的过程中,VEGF蛋白表达明显增加、PEDF蛋白表达明显下降(P＜0.05)。
3.凉血化瘀方治疗CNV组,FFA检查测量荧光渗漏面积可见3w～4w时,中药组CNV的荧光渗漏面积明显小于空白组和对照组(P＜0.05);脉络膜铺片新生血管面积测量可见4w时CNV面积明显小于空白组和对照组(P＜0.05);FⅧR:Ag因子和VEGF蛋白表达可见4w时中药组阳性反应物IOD值小于空白组和对照组(P＜0.05),而PEDF蛋白表达在1w～4w时则明显增加(P＜0.05)。
4.凉血化瘀方治疗湿性AMD患者3月后,视力提高25眼(22.94%),稳定74眼(67.89%),降低10眼(9.17%);治疗6月后,视力提高32眼(29.36%),稳定60眼(55.05%),降低17眼(15.60%),低视力组患者视力提高和稳定程度均好于高视力组。治疗3、6个月后患者眼底出血面积、渗出面积、累积损害面积、黄斑视网膜厚度、黄斑区总容积改变无统计学意义。
结论:
1.激光诱导的BN大鼠CNV模型,与人类AMD的CqqV在发生、发展和病理基础上基本一致,是目前研究湿性年龄相关性黄斑变性的良好模型。
2.FFA检查可确定CNV的生成,通过对CNV渗漏面积的统计,可半定量估算新生血管的生长情况。FITC-D血管灌注的脉络膜铺片法可保留CNV的原始形态,具有易观察性,并可做客观的定量统计,是研究CNV的可靠方法。FⅧR:Ag可标记血管内皮细胞,是判定CNV存在的有效方法。
3.实验性CNV中VEGF的表达上调、PEDF表达下降,二者呈明显负相关关系。
4.通过脉络膜血管铺片测量CNV面积、FFA检查、病理、免疫组化检查可观察到,凉血化瘀方可以显著抑制实验性脉络膜新生血管的生长,下调VEGF、上调PEDF蛋白表达水平。
5.凉血化瘀方治疗湿性AMD患者在6个月观察期内具有一定的保持视力稳定和提高作用,能使眼底出血、渗出、累积损害和水肿瘢痕情况稳定;不良事件发生率低;是治疗湿性AMD患者的安全有效方法。
Objective:
1.To study the result of FFA,choroidal tissue and expression of FⅧR:Ag in choroidal neovascularization(CNV),to investigate the progress and characters of CNV induced by krypton laser in BN rat.
2.To study the expression and the relationship of vascular endothelial growth factor(VEGF) and pigment epithelium-derived factor(PEDF) in choroidal neovascularization.
3.To study the inhibition of experimental CNV by Decoction of Liangxue Huayu
4.To study the clinical therapeutic effect of age-related macular degeneration(AMD) by Decoction of Liangxue Huayu.
Methods:
1.Four groups of 24 BN rats were photocoagulated with 20 spots by krypton laser in single eye.1 weeks,2 weeks,3 weeks,4 weeks after photocoagulation,photo of eye ground and FFA examination were performed,the quantities of CNV was measured by choroidal tissue analysis in 2 BN rats every week.The other 4 rats were executed with eyes excising,paraffin sections were made by these samples and observing the pathology and expression of FⅧR:Ag.
2.Study the growth of CNV in four weeks and the expression of VEGF and PEDF in it.
3.Three groups of 48 BN rats were photocoagulated by krypton laser in both eyes. One day after photocoagulation,one group of 16 rats received intragastric administration of Decoction of Liangxue Huayu,and the other group intravitreal injection of 2μl triamcinolone as control group.1 weeks to 4 weeks after photocoagulation,FFA was performed first.1 rat was vaso-infused with fluorescein isothiocyanate dextran(FITC-D) and the area of CNV was estimated.Other samples were made to paraffin sections and estimated the expression of FⅧR:Ag,VEGF and PEDE
4.Study the therapeutic effect of 109 eyes in 90 AMD patients by Decoction of Liangxue Huayu in 6 months.Analysis the results of eye sight,area of exudation and hemorrhage,area of total affected,foveal thickness and total macular volume and evaluate the untoward reaction.
Results:
1.FFA,measurement of CNV area by stretched preparation of choroid and pathology, expression of FⅧR:Ag showed the increasing area of experimental CNV in 4 weeks (P＜0.05).
2.The expression of VEGF increasing and PEDF decreasing in the formation of experimental CNV(P＜0.05).
3.FFA and stretched preparation of choroid for quantitative analysis of CNV showed fewer area of CNV than blank and control groups at 4 weeks(P＜0.05).While the expression of FⅧR:Ag and VEGF were fewer than blank and control groups at 4 weeks(P＜0.05),the expression of PEDF was higher than those groups from 1 week to 4 weeks(P＜0.05).
4.After 3 months treating AMD patients by Decoction of Liangxue Huayu,eye sight increased in 25 eyes(22.94%),stabled in 74 eyes(67.89%) and decreased in 10 eyes(9.17%); after 6 months,eye sight increased in 32 eyes(29.36%),stabled in 60 eyes(55.05%) and decreased in 17 eyes(15.6%).And there is no statistical meaning with the area of hemorrhage and exudation,area of total affected,foveal thickness and total macular volume.
Conclusion:
1.Krypton laser photocoagulation inducing CNV of BN rat may use for studying on AMD because its similarity in the growth,pathology with human's CNV.
2.FFA examination,immunohustochemistry of FⅧR:Ag and stretched preparation of choroid for half-quantitative analysis of CNV were reliable index estimating CNV.
3.Mechanism of CNV formation involves the balance of the expression of VEGF and PEDF.
4.Decoction of Liangxue Huayu can inhibit the growth of experimental CNV through decreasing the expression of VEGF and increasing the expression of PEDF.
5.Decoction of Liangxue Huayu can increased or stable most eye sight of exudation AMD patients,stabilization edema of macula and the area of hemorrhage and exudation, area of total affected with few untoward reaction.It's a safe and effective therapy.
1.评价激光诱导棕色挪威(brown norway,BN)大鼠脉络膜新生血管(choroidalneovascularization,CNV)模型的可行性,并从荧光素眼底血管造影(fundus fluoresceinangiography,FFA)、脉络膜铺片血管定量分析、病理、血管内皮细胞免染色疫组化法进行多方面比较。
2.观察评价实验性脉络膜新生血管中血管内皮生长因子(vascular endothelialgrowth factor,VEGF)与色素上皮衍生因子(pigment epithelium-derived factor,PEDF)的表达水平及相互关系。
3.观察凉血化瘀方对激光诱导的BN大鼠CNV形成的抑制作用,从FFA检查、脉络膜铺片血管定量分析、病理、血管内皮细胞染色免疫组化法、VEGF与PEDF蛋白表达水平方面进行多方位评价。
4.观察凉血化瘀方治疗湿性年龄相关性黄斑变性(age-related macular degeneration,AMD)患者的临床疗效,评价指标有:视力、FFA检查、眼底出血面积、渗出面积、累积损害面积、黄斑视网膜厚度、黄斑区总容积。
方法:
1.对4组24只BN大鼠单眼进行氪激光视网膜光凝,围绕视盘等距光凝20个激光斑,诱导CNV形成。激光功率360mW,光斑直径50μm,曝光时间0.05s。分别在光凝后1w、2w、3w、4w进行眼底彩照和FFA检查。在上述时间点各取2只大鼠进行脉络膜铺片定量分析血管面积;余4只处死,取眼球做石蜡切片,观察激光斑病理改变,FⅧR:Ag(FactorⅧ-Related Antigen,Ⅷ因子相关抗原)免疫组化法对新生血管进行半定量分析。
2.通过石蜡切片观察CNV发展的4w时间内,不同时间点CNV中细胞因子VEGF和PEDF的蛋白表达。
3.对3组48只BN大鼠行氪激光光凝,建立CNV动物模型,其中1组作为空白组,1组造模后玻璃体腔注射曲安奈德做为对照组,1组造模后予以凉血化瘀方灌胃作为中药组。分别在光凝后1w、2w、3w、4w行FFA检查。在上述各时间点处死各组动物4只,其中1只行异硫氰酸荧光素葡聚糖(Fluorescein isothiocyanate dextran,FITC-D)灌注、脉络膜铺片定量新生血管面积,余3只摘出眼球,制作石蜡切片,观察不同时间点CNV的中FⅧR:Ag、VEGF、PEDF表达的变化,并进行半定量检测。
4.观察临床确诊为CNV型AMD的患者90例患者109眼,予以口服凉血化瘀中药治疗。连续观察6个月,总结视力,眼底出血面积、渗出面积、累积损害面积、OCT检查结果。分析比较治疗前后视力、眼底出血、渗出、累积损害面积、黄斑视网膜厚度和黄斑区总容积变化,并观察用药过程中不良反应。
结果:
1.在4w时间内,FFA检查、脉络膜铺片血管面积测量、病理切片、FⅧR:Ag因子免疫组化结果显示,实验性CNV在1w～3w期间呈持续性增殖(P＜0.05),到4w时稳定。
2.在实验性CNV形成的过程中,VEGF蛋白表达明显增加、PEDF蛋白表达明显下降(P＜0.05)。
3.凉血化瘀方治疗CNV组,FFA检查测量荧光渗漏面积可见3w～4w时,中药组CNV的荧光渗漏面积明显小于空白组和对照组(P＜0.05);脉络膜铺片新生血管面积测量可见4w时CNV面积明显小于空白组和对照组(P＜0.05);FⅧR:Ag因子和VEGF蛋白表达可见4w时中药组阳性反应物IOD值小于空白组和对照组(P＜0.05),而PEDF蛋白表达在1w～4w时则明显增加(P＜0.05)。
4.凉血化瘀方治疗湿性AMD患者3月后,视力提高25眼(22.94%),稳定74眼(67.89%),降低10眼(9.17%);治疗6月后,视力提高32眼(29.36%),稳定60眼(55.05%),降低17眼(15.60%),低视力组患者视力提高和稳定程度均好于高视力组。治疗3、6个月后患者眼底出血面积、渗出面积、累积损害面积、黄斑视网膜厚度、黄斑区总容积改变无统计学意义。
结论:
1.激光诱导的BN大鼠CNV模型,与人类AMD的CqqV在发生、发展和病理基础上基本一致,是目前研究湿性年龄相关性黄斑变性的良好模型。
2.FFA检查可确定CNV的生成,通过对CNV渗漏面积的统计,可半定量估算新生血管的生长情况。FITC-D血管灌注的脉络膜铺片法可保留CNV的原始形态,具有易观察性,并可做客观的定量统计,是研究CNV的可靠方法。FⅧR:Ag可标记血管内皮细胞,是判定CNV存在的有效方法。
3.实验性CNV中VEGF的表达上调、PEDF表达下降,二者呈明显负相关关系。
4.通过脉络膜血管铺片测量CNV面积、FFA检查、病理、免疫组化检查可观察到,凉血化瘀方可以显著抑制实验性脉络膜新生血管的生长,下调VEGF、上调PEDF蛋白表达水平。
5.凉血化瘀方治疗湿性AMD患者在6个月观察期内具有一定的保持视力稳定和提高作用,能使眼底出血、渗出、累积损害和水肿瘢痕情况稳定;不良事件发生率低;是治疗湿性AMD患者的安全有效方法。
Objective:
1.To study the result of FFA,choroidal tissue and expression of FⅧR:Ag in choroidal neovascularization(CNV),to investigate the progress and characters of CNV induced by krypton laser in BN rat.
2.To study the expression and the relationship of vascular endothelial growth factor(VEGF) and pigment epithelium-derived factor(PEDF) in choroidal neovascularization.
3.To study the inhibition of experimental CNV by Decoction of Liangxue Huayu
4.To study the clinical therapeutic effect of age-related macular degeneration(AMD) by Decoction of Liangxue Huayu.
Methods:
1.Four groups of 24 BN rats were photocoagulated with 20 spots by krypton laser in single eye.1 weeks,2 weeks,3 weeks,4 weeks after photocoagulation,photo of eye ground and FFA examination were performed,the quantities of CNV was measured by choroidal tissue analysis in 2 BN rats every week.The other 4 rats were executed with eyes excising,paraffin sections were made by these samples and observing the pathology and expression of FⅧR:Ag.
2.Study the growth of CNV in four weeks and the expression of VEGF and PEDF in it.
3.Three groups of 48 BN rats were photocoagulated by krypton laser in both eyes. One day after photocoagulation,one group of 16 rats received intragastric administration of Decoction of Liangxue Huayu,and the other group intravitreal injection of 2μl triamcinolone as control group.1 weeks to 4 weeks after photocoagulation,FFA was performed first.1 rat was vaso-infused with fluorescein isothiocyanate dextran(FITC-D) and the area of CNV was estimated.Other samples were made to paraffin sections and estimated the expression of FⅧR:Ag,VEGF and PEDE
4.Study the therapeutic effect of 109 eyes in 90 AMD patients by Decoction of Liangxue Huayu in 6 months.Analysis the results of eye sight,area of exudation and hemorrhage,area of total affected,foveal thickness and total macular volume and evaluate the untoward reaction.
Results:
1.FFA,measurement of CNV area by stretched preparation of choroid and pathology, expression of FⅧR:Ag showed the increasing area of experimental CNV in 4 weeks (P＜0.05).
2.The expression of VEGF increasing and PEDF decreasing in the formation of experimental CNV(P＜0.05).
3.FFA and stretched preparation of choroid for quantitative analysis of CNV showed fewer area of CNV than blank and control groups at 4 weeks(P＜0.05).While the expression of FⅧR:Ag and VEGF were fewer than blank and control groups at 4 weeks(P＜0.05),the expression of PEDF was higher than those groups from 1 week to 4 weeks(P＜0.05).
4.After 3 months treating AMD patients by Decoction of Liangxue Huayu,eye sight increased in 25 eyes(22.94%),stabled in 74 eyes(67.89%) and decreased in 10 eyes(9.17%); after 6 months,eye sight increased in 32 eyes(29.36%),stabled in 60 eyes(55.05%) and decreased in 17 eyes(15.6%).And there is no statistical meaning with the area of hemorrhage and exudation,area of total affected,foveal thickness and total macular volume.
Conclusion:
1.Krypton laser photocoagulation inducing CNV of BN rat may use for studying on AMD because its similarity in the growth,pathology with human's CNV.
2.FFA examination,immunohustochemistry of FⅧR:Ag and stretched preparation of choroid for half-quantitative analysis of CNV were reliable index estimating CNV.
3.Mechanism of CNV formation involves the balance of the expression of VEGF and PEDF.
4.Decoction of Liangxue Huayu can inhibit the growth of experimental CNV through decreasing the expression of VEGF and increasing the expression of PEDF.
5.Decoction of Liangxue Huayu can increased or stable most eye sight of exudation AMD patients,stabilization edema of macula and the area of hemorrhage and exudation, area of total affected with few untoward reaction.It's a safe and effective therapy.
引文
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