CN118059213A - Pd-1在制备治疗眼部脉络膜新生血管药物中的应用 - Google Patents
Pd-1在制备治疗眼部脉络膜新生血管药物中的应用 Download PDFInfo
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Abstract
本发明公开了PD‑1在制备治疗眼部脉络膜新生血管药物中的应用,所述的应用为抑制眼部视网膜神经炎症,抑制眼内新生血管的渗漏,可辅助治疗新生血管性年龄相关性黄斑变性。
Description
技术领域
本发明具体涉及PD-1新用途技术领域,具体涉及PD-1相关制剂在制备治疗眼部新生血管药物中的应用。
背景技术
新生血管性年龄相关性黄斑变性是引起老年人口不可逆视力丧失的主要原因。其病变特征为眼部病理性新生血管,会引起严重的视力丧失甚至失明。
目前主要的治疗方法为抗VEGF药物,虽然有较好的疗效,但是仍有部分患者对治疗不敏感。而且抗VEGF需要反复多次注射,可能会引起白内障,青光眼,眼内炎等并发症。对患者经济及身体造成负担。此外,VEGF有神经营养作用,如果使用过度,可能会对神经元造成损伤。
PD-1(Programmed death protein 1)程序性细胞死亡蛋白受体1,是Ⅰ型跨膜受体,属于免疫球蛋白超家族。在胸腺细胞、外周CD4+、CD8+、NK T细胞、B细胞、巨噬细胞和树突状细胞上表达。是重要的免疫检查点分子,在免疫监视,免疫赦免等方面发挥重要作用。在肿瘤治疗领域,阻断PD-1治疗发挥了重要作用。阻断PD-1可能会加重中枢神经系统炎症。PD-1对视网膜神经炎症的治疗作用研究较少,目前尚无采用PD-1治疗新生血管的报道。
本发明探索PD-1对小胶质细胞的调控可作为新生血管治疗的新方向。
发明内容
为了解决现有技术存在的技术缺陷,本发明提供了PD-1作为制备治疗眼部脉络膜新生血管药物的应用,通过抑制小胶质细胞过度激活找到一种抑制眼内新生血管的方法。
所述的治疗眼部脉络膜新生血管为抑制眼部视网膜神经炎症,抑制抑制眼内新生血管的渗漏。
所述的药物包括药学上有效量的PD-1和药学上可接受的载体。
所述的载体选自常用的药用辅料、生理盐水或蒸馏水。
上述用于治疗眼部脉络膜新生血管药物,所述的药物以PD-1为有效成分,向PD-1中加入常规辅料按照常规工艺制成临床上可接受的合剂、胶囊剂、片剂、薄膜剂、滴眼液、注射剂。
进一步的,本发明药物应用于治疗病理性眼内新生血管。
与现有技术比较本发明的有益效果:
本发明提供了PD-1相关制剂治疗眼部新生血管的药物应用,可抑制眼部视网膜神经炎症,抑制眼内新生血管的渗漏。
附图说明
图1为PD-1对激光诱导小鼠新生血管的治疗作用图。
图1-A为实施例1玻璃体腔注射PD-1后对视网膜的光学相干断层扫描图(OCT),眼底荧光素血管造影图(FFA),脉络膜铺片IB4免疫荧光染色图(IF)。
图1-A左侧列:OCT检测眼球内注射PD-1和溶剂组眼内新生血管。图1-A中间列:FFA显示球内注射PD-1和溶剂组眼内新生血管渗漏情况。图1-A右侧列:脉络膜铺片IB4染色对比PD-1和溶剂组眼内新生血管。
图1-B为OCT测量两组CNV高度值统计图。
图1-C为PD-1和溶剂组CNV平均渗漏等级统计图。
图1-D为IB4染色测量PD-1和溶剂组CNV面积值统计图。
图2为PD-1对激光诱导小鼠视网膜神经炎症的抑制作用图。
图中,A-E是与对照组相比,激光造模后球内注射溶剂和PD-1组对视网膜炎症因子Cd68,Ccl-2,Il1b,Il6,Tnf mRNA水平的作用。
具体实施方式
以下通过实施例对本发明进行进一步解释说明:PD-1,购买自ABclone公司。
实施例1
1.小鼠腹腔注射1%戊巴比妥钠(40mg/kg)麻醉,使用0.5%托吡卡胺滴眼液扩张瞳孔,激光光凝术采用裂隙灯式二极管激光系统,绿光波长为532nm,功率为100mW,持续时间为100ms,光斑尺寸为100μm。在视神经周围应用4个激光点。用8-10周龄的C57BL/6小鼠在激光诱导新生血管造模后玻璃体注射PD-1,(PD-1蛋白购买自ABclone公司,生理盐水稀释为5ng/μl),在实验7天后行FFA以及OCT检查结果显示渗漏明显减少。实验结果见图1,图中A左侧列:OCT检测眼球内注射PD-1和溶剂组眼内新生血管。A中间列:FFA显示球内注射PD-1和溶剂组眼内新生血管渗漏情况。A右侧列:脉络膜铺片IB4染色对比PD-1和溶剂组眼内新生血管面积。
由图1的脉络膜铺片结果显示药物处理组用激光诱导出的眼内新生血管与生理盐水溶剂对照组相比明显变小。说明PD-1对小鼠新生血管模型中的新生血管有明显的抑制效果。
2.玻璃体内注射,小鼠被麻醉并使用上述方法扩大其瞳孔。使用30.5号针头(Becton-Dickinson,Houston,TX),在鼻上缘后方立即穿过结膜和巩膜进行初步穿刺。随后,将32号钝针连接5μl注射器(Hamilton,Reno,NV)通过该切口插入玻璃体腔。标准进样量为1μL。分为以下几组:PD-1(5ng)和载体(仅限生理盐水)。
3.小鼠麻醉并散瞳行眼底荧光素血管造影(FFA)检查时,将光源过渡到蓝色通道,并仔细校准光强。随后,腹腔注射2%荧光素钠溶液1.7ml/kg。随后拍摄的照片记录了FFA图像。对每只实验动物的双眼进行拍摄,确保数据收集的全面。实验后的步骤包括用生理盐水冲洗眼睛,并使用左氧氟沙星滴眼液预防潜在感染。根据拍摄图片(图1)确定渗漏点分级1级,表示“无泄漏”,存在微弱的高荧光或斑点荧光;2级,被称为“可疑渗漏”,表明晚期高荧光没有大小或强度增加;3级,称为“渗漏”,描述了高荧光区域内的荧光强度增加,而大小保持不变的情况;4级,被称为“病理上显著的渗漏”,其特征是荧光强度和大小都增加载体组平均等级2.1,PD-1组平均等级1.3.
4.光学相干断层扫描(OCT)检查:将固定并麻醉好的动物置于升降桌上,使用复方托吡卡胺滴眼液常规散瞳,并用盐酸丙美卡因滴眼液进行表麻,摆好眼位,待瞳孔散大后将医用卡波姆滴眼液凝胶涂于待测眼角膜上,将眼科超显微成像系统光源调整聚焦,调整镜头焦点至视网膜,而后对视网膜进行光学相干断层扫描(OCT)。每只实验动物双眼均进行拍照。实验完成后用生理盐水清洗双眼,并用左氧氟沙星滴眼液滴眼预防感染。ImageJ软件测量视网膜下新生血管的高度,单位(μm)。载体组平均高度37.58μm,PD-1组平均高度53.50μm(图1.B)。
5.采用颈椎脱位法对小鼠实施安乐死,取下眼球,用4%多聚甲醛(PFA)固定2小时。分离视网膜和RPE-脉络膜复合体,进行通透和封闭。随后,将一抗(抗iba1)在4℃下孵育24小时。一抗孵育后,样品随后与二抗山羊抗兔AlexaFluorTM 647(1:1000稀释)孵育2小时。对于RPE/脉络膜样本,使用IB4(1:500稀释,Sigma-Aldrich)进行染色。经过三次PBS洗涤后,将视网膜和RPE/脉络膜样本小心地平放在玻璃显微镜载玻片上进行成像。采用ImageJ软件进行盲法图像分析,定量评估激光病变面积,载体组平均面积21209μm2,PD-1组平均面积37946μm2(图1.C)。
6.小鼠视网膜样本取出后立即快速冷冻。总RNA提取采用美国EZBioscience公司组织RNA纯化试剂盒。随后,利用EZscript Reverse Transcription Mix II withgDNARemover(EZBioscience,USA)合成cDNA。采用Bio-Rad RT-PCR系统(Hercules,USA)和2×SYBR Green qPCR Master Mix进行基因表达分析。β-肌动蛋白作为内参。用2^-ΔΔCt方法定量测定基因表达的相对变化。每个样本都进行了三次分析,所提供的数据代表了三次独立评估的平均值(见图2)。
上述实验结果显示,PD-1眼内注射后,新生血管面积、高度均较对照组减轻,并且PD-1可以抑制激光诱导上调的视网膜炎症因子mRNA水平。根据上述实验,证明PD-1可抑制视网膜炎症,从而抑制眼内新生血管,可辅助治疗湿性黄斑病变。
Claims (6)
1.PD-1在制备治疗眼部脉络膜新生血管药物中的应用。
2.根据权利要求1所述的应用,其特征在于所述的治疗眼部脉络膜新生血管为抑制眼部视网膜神经炎症,抑制眼内新生血管的渗漏。
3.根据权利要求1所述的应用,其特征在于所述的药物包括药学上有效量的PD-1和药学上可接受的载体。
4.根据权利要求3所述的应用,其特征在于所述的载体选自常用的药用辅料、生理盐水或蒸馏水。
5.一种用于治疗眼部脉络膜新生血管的药物,所述的药物以PD-1为有效成分,向PD-1中加入常规辅料按照常规工艺制成临床上可接受的制剂。
6.根据权利要求5所述的药物,其特征在于所述的制剂为滴眼液、注射剂、合剂、胶囊剂、片剂、薄膜剂。
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