黄斑变性1号方治疗渗出型年龄相关性黄斑变性的临床观察及机制研究

发布时间：2018-05-04 21:08

本文选题：雷珠单抗 + 渗出型年龄相关性黄斑变性　；参考：《北京中医药大学》2017年博士论文

【摘要】：(一)黄斑变性1号方治疗渗出型年龄相关性黄斑变性的临床观察研究目的:观察黄斑变性1号方(Huangban Bianxing One Decoction,HBOD)联合雷珠单抗治疗渗出型年龄相关性黄斑变性(aged-related macular degeneration,AMD)患者的有效性和安全性。方法:从2014年7月至2016年2月,共75例符合纳入标准和排除标准的渗出型AMD患者(75只眼)入选本次研究,随机分为治疗组和对照组,两组均在入组后给予雷珠单抗治疗1次。在此基础上,治疗组每天口服HB0D水煎剂,1天2次,每次100mL,疗程持续6个月。作为主要结果,评估治疗前后的早期治疗糖尿病视网膜病变研究(early treatment diabetic retinopathy study,ETDRS)字母数的最佳矫正视力、黄斑中心凹厚度(center macular thickness,CMT)、病变高度、眼底出血面积、眼底荧光渗漏面积。结果:经过3个月的治疗,两组的ETDRS字母数都获得了较大改善(P0.05)。治疗6个月后,治疗组的ETDRS字母数明显提高(P0.01),且明显优于对照组(P0.05)。治疗3个月后,两组相比于治疗前CMT和病变高度也明显降低(P0.01或P0.05),治疗6个月后,治疗组相比治疗前CMT和病变高度也明显降低(P0.01)。治疗6个月后,出血面积和荧光素渗漏面积也明显缩小(P0.01或P0.05),且与对照组比较,治疗后的出血面积和荧光渗漏面积减小明显优于对照组(P0.01或P0.05)。治疗期间,也未有明确与治疗有关的重大不良事件。结论:渗出型AMD属于新生血管性视网膜病变,是世界老年人口失明的主要原因之一。在本研究结果的基础上,HBOD可以辅助改善视力,并减少渗出型AMD患者的出血和荧光素渗漏,减少抗VEGF药物注射次数,这可能是渗出型AMD有效的辅助和安全治疗方法。(二)黄斑变性1号方治疗渗出型年龄相关性黄斑变性的机制研究目的:围绕白细胞介素(interleukin,IL)-17信号调控通路,探讨中药复方HBOD对渗出型AMD患者血浆中IL-17、血管内皮生长因子(vascular endothelial growth factor,VEGF)-A表达水平的影响。方法:2014年7月至2016年2月,纳入临床观察研究中治疗组的38例渗出型AMD患者。治疗前和服用中药复方HBOD 6个月后患者于清晨空腹抽取静脉血,分别作为AMD组和治疗组,并招募20例健康者作为对照组。并用酶联免疫吸附测定法测定血浆中IL-17A、VEGF-A 表达水平。结果:本次研究中,IL-17A在对照组中仅有1例可测出,AMD组中有6例,治疗组中有3例,因而无法做统计学差异比较。AMD组血浆中的VEGF-A水平明显高于治疗组和对照组(P＜0.01或P0.05),治疗后与对照组比较无显著性差异(P0.05)。结论:HB0D可以调节渗出型AMD患者血浆中的VEGF-A的水平,但HBOD是否能影响IL-17A的水平还有待进一步研究。(三)黄斑变性1号方治疗渗出型年龄相关性黄斑变性的血浆代谢组学研究目的:AMD是具有高发病率和复杂发病机制的主要失明疾病。我们的目的是研究中药复方HBOD治疗渗出型AMD患者血浆代谢组学特征。方法:这项代谢组学研究是通过分析20例健康对照者和20例渗出型AMD患者接受HBOD治疗前与治疗6个月后的的血浆进行的。我们使用超高压液相色谱和四极杆飞行时间质谱法进行分析。这些差异与AMD病理生理和治疗的关系也得到了确定。剩余数据通过Pareto scaling进行归一化,然后通过使用MetaboAnalysis软件的多变量数据分析处理有效数据,包括无监督主成分分析(principal component analysis,PCA)和监督偏最小二乘法分析(partial least squares-discriminate analysis,PLS-DA)。目的是确定分析的重要代谢物。进行分层聚类,以区分组间的代谢物。然后使用建立的数据库鉴定重要的代谢物,并将其进行京都基因和基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)代谢通路分析。结果:PCA、PLS-DA和分层聚类结果分析显示,渗出型AMD治疗前后血浆代谢状态无明显变化。在正离子模式下,检测到总共5443个离子峰,其中包括一些氨基酸、异麦芽糖、氢化可的松和胆红素10种代谢物在渗出型AMD和健康对照之间有显著不同。氨基酸生物合成的代谢通路在渗出型AMD患者和对照者间有显著差异。结论:这些研究结果表明,渗出型AMD和对照之间的代谢特征不同,并且可能为AMD指导的治疗和诊断提供有希望的新目标。同时,因建立的模型可靠性较差,尚未发现HBOD可以显著调节渗出型AMD患者的代谢状态。(四)黄斑变性1号方及其主要成分干预IL-17A刺激ARPE-19细胞分泌促炎因子研究目的:本研究旨在探讨中药复方HBOD及其主要成分对IL-17A在人视网膜上皮细胞ARPE-19细胞中诱导促炎细胞因子产生的影响。方法：将 ARPE-19 细胞与 100ng/mL IL-17A 孵育 6、12、24 或 36h,以及与 10、20、50、100或200ng/mL IL-17A孵育6h,通过定量实时聚合酶链反应(quantitative real-time polymerase chain reaction,qRT-PCR)以测定IL-1 β、IL-6、IL-8、趋化因子(C-C 形)配体(chemokine(C-Cmotif)ligands,CCLs)CCL2 和 CCL20mRNA 的表达。进行细胞增殖-毒性检测试剂盒(Cell Counting Kit,CCK-8)测定以确定HB0D、黄芪多糖(astragalus polysacharin,APS),三七总皂甙(panax notoginseng saponins,PNS)和贝母素甲(peimine,PE)的细胞毒性。ARPE-19细胞用非细胞毒性药物溶液预处理2h,然后给与或不与人重组IL-17A孵育6h或24h。分别通过qRT-PCR和酶联免疫吸附测定法测定所得的促炎细胞因子mRNA和蛋白表达水平。结果:IL-17A 可显著增加 ARPE-19 细胞中的 IL-1 β、IL-6、IL-8、CCL2 和 CCL20mRNA和蛋白表达(P0.01)。HB0D可以抑制上述促炎细胞因子mRNA和蛋白表达水平(P0.01或P0.05)。作为主要成分,APS、PNS和PE能部分或全部抑制这些促炎细胞因子mRNA和蛋白表达水平(P0.01)。且PE对CCL20表达有很强的抑制作用,其抗炎作用更广泛。我们的研究结果表明,HB0D及其主要成分可能通过部分抑制由IL-17A诱导的视网膜色素上皮细胞的炎症反应来治疗AMD。结论:对IL-17A诱导的促炎细胞因子产生的抑制作用可能解释了 HB0D治疗AMD的有效机制。
[Abstract]:(1) a clinical study of macular degeneration 1 in the treatment of exudative age-related macular degeneration. Objective: To observe the efficacy and safety of Huangban Bianxing One Decoction (HBOD) combined with lezumab in the treatment of exudative age-related macular degeneration (aged-related macular degeneration, AMD). Methods: from 20 From July to February 2016 14 years, 75 cases of exudative AMD patients (75 eyes) were enrolled in this study. They were randomly divided into treatment group and control group. The two groups were treated with leuzumab for 1 times after entering group. On this basis, the treatment group took HB0D Decoction every day, 1 days 2 times, each time lasted for 6 months. The main results were to evaluate the best corrected visual acuity of the early treatment of diabetic retinopathy (early treatment diabetic retinopathy study, ETDRS), the thickness of the macular fovea (center macular thickness, CMT), the height of the lesion, the area of the fundus hemorrhage, and the fluorescent leakage area of the fundus. Results: after 3 months of treatment, two The number of ETDRS letters in the group was greatly improved (P0.05). After 6 months of treatment, the number of ETDRS letters in the treatment group was significantly increased (P0.01), and obviously superior to the control group (P0.05). After 3 months of treatment, the two groups were also significantly lower (P0.01 or P0.05) than before the treatment and the height of the lesion (P0.01 or P0.05). After 6 months of treatment, the treatment group compared with the pre treatment CMT and the height of the lesion. Obviously decreased (P0.01). After 6 months of treatment, the area of hemorrhage and the area of fluorescein leakage were also significantly reduced (P0.01 or P0.05), and compared with the control group, the area of hemorrhage and the area of fluorescent leakage were significantly lower than those of the control group (P0.01 or P0.05). During the treatment, there were no significant adverse events related to the treatment. Conclusion: exudative AMD Neovascular retinopathy is one of the main causes of blindness in the aged in the world. On the basis of this study, HBOD can help improve vision, reduce bleeding and fluorescein leakage in exudative AMD patients, and reduce the number of anti VEGF drug injections. This may be an effective adjuvant and safe treatment for exudative AMD. (two) Study on the mechanism of macular degeneration 1 in the treatment of exudative age-related macular degeneration. Objective: To explore the effect of traditional Chinese medicine compound HBOD on the level of IL-17, vascular endothelial growth factor (vascular endothelial growth factor, VEGF) -A expression in plasma of exudative AMD patients by interleukin (IL) -17 signal regulation pathway. Method: 201 From July to February 2016 4 years, 38 cases of exudative AMD patients in the clinical observation group were included in the treatment group. Before and 6 months after the treatment of the compound HBOD, the patients were selected as the AMD group and the treatment group, and 20 healthy persons were recruited as the control group, and the plasma IL-17A, VEGF-A was measured by the enzyme immunoadsorption assay. Results: in this study, there were only 1 cases of IL-17A in the control group, 6 in group AMD and 3 in the treatment group, and there were 3 cases in the treatment group, so the VEGF-A level in the plasma of.AMD group was significantly higher than that in the treatment group and the control group (P < 0.01 or P0.05). There was no significant difference between the control group and the control group (P0.05). Conclusion HB0D The level of VEGF-A in plasma of exudative AMD patients can be regulated, but whether HBOD can affect the level of IL-17A remains to be further studied. (three) the objective of plasma metabonomics for the treatment of exudative age related macular degeneration by macular degeneration 1 Prescription: AMD is a major blindness disease with high incidence and complex pathogenesis. Our objective We studied the plasma metabolic characteristics of the HBOD patients with exudative AMD. Methods: This metabonomics study was conducted by analyzing 20 healthy controls and 20 cases of exudative AMD patients before and after 6 months of treatment with HBOD. We used high pressure liquid chromatography and quadrupole time of flight mass spectrometry. The relationship between these differences and the pathophysiology and treatment of AMD is also determined. The remaining data are normalized by Pareto scaling, and then the effective data are processed by multivariable data analysis using MetaboAnalysis software, including unsupervised principal component analysis (principal component analysis, PCA), and supervised partial least square method. Analysis (partial least squares-discriminate analysis, PLS-DA). The purpose is to determine the important metabolites of the analysis. Stratify clustering to distinguish between groups of metabolites. Then use the established database to identify important metabolites and carry out the Kyoto gene and genome 100 family (Kyoto Encyclopedia of Genes and Genomes, KEGG). Results of metabolic pathway analysis. Results: PCA, PLS-DA and stratified cluster analysis showed that there was no significant change in plasma metabolic state before and after the exudative AMD treatment. In the positive ion mode, a total of 5443 ion peaks were detected, including some amino acids, ISO maltose, hydrocortisone and bilirubin 10 metabolites in the exudative AMD and health control There were significant differences between the metabolic pathways of the amino acid biosynthesis in the exudative AMD patients and the controls. Conclusions: These findings suggest that the metabolic characteristics between the exudative AMD and the control are different, and may provide promising new targets for the treatment and diagnosis of AMD guidance. It is not found that HBOD can significantly regulate the metabolic state of exudative AMD patients. (four) macular degeneration No. 1 and its main components interfere with IL-17A stimulation of ARPE-19 cells to secrete proinflammatory cytokines. The purpose of this study was to explore the effect of HBOD and its main components on the induction of proinflammatory cells in the ARPE-19 cells of human retina membrane epithelial cells. Methods: to incubate ARPE-19 cells with 100ng/mL IL-17A for 6,12,24 or 36h, and to incubate 6h with 10,20,50100 or 200ng/mL IL-17A, by quantitative real-time polymerase chain reaction (quantitative real-time polymerase) (C-Cmotif) ligands, CCLs) expression of CCL2 and CCL20mRNA. Cell proliferation toxicity detection kit (Cell Counting Kit, CCK-8) was used to determine HB0D, astragalus polysaccharide (Astragalus Polysacharin,), 37 total saponins and fritillin a cytotoxic cells used for non cytotoxicity. Pretreated 2h, and then incubated with or without human recombinant IL-17A to incubate 6h or 24h. by qRT-PCR and enzyme linked immunosorbent assay to determine the level of proinflammatory cytokines mRNA and protein expression. Results: IL-17A can significantly increase IL-1 beta, IL-6, IL-8, CCL2, CCL2 and protein expression in ARPE-19 cells. D can inhibit the above proinflammatory cytokine mRNA and protein expression level (P0.01 or P0.05). As a major component, APS, PNS and PE can partially or completely inhibit the level of mRNA and protein expression (P0.01) of these proinflammatory cytokines. And PE has a strong inhibitory effect on CCL20 expression, and its anti-inflammatory effect is more extensive. Our results showed that HB0D and The main components may be treated by partially inhibiting the inflammatory response of IL-17A induced retinal pigment epithelial cells to treat the AMD. conclusion: the inhibitory effect of IL-17A induced proinflammatory cytokines may explain the effective mechanism of HB0D for the treatment of AMD.

【学位授予单位】：北京中医药大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R774.5

【参考文献】