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2019年血源性滴眼液治疗重度干眼专家共识解读

Interpretation of expert consensus on the treatment of severe dry eye with blood-based eye drops in 2019

来源期刊: 眼科学报 | 2021年4月 第36卷 第4期 293-298 发布时间: 收稿时间:2023/5/11 17:27:43 阅读量:4762
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关键词:
干眼血源性滴眼液治疗
dry eye blood-based eye drop treatment
DOI:
10.3978/j.issn.1000-4432.2021.03.07
血源性滴眼液是治疗干眼的一种有效的治疗方法。来源可分为自体、供体,其中供体来源又可分为成人外周血及脐带血,并以血清、富血小板血浆、血小板来源生长因子以及血小板裂解产物的形式制成。由于各种血液制品的原理及适应症不同,本文将对不同血源性滴眼液制品的制备及面临的挑战的共识问题进行解读。
Blood-based eye drops can be used effectively in the treatment of dry eyes. The sources of blood-based eye drops can be divided into autologous and donor, and donor sources can be divided into adult peripheral blood and umbilical cord blood, which are prepared in the form of serum, platelet-rich plasma, platelet-derived growth facters and platelet lysate. Due to the different principles and indications of various blood products, the consensus issues on the preparation and challenges of different blood-based eye drop products are reviewed.
干眼(dry eye,DE)是眼表的一种多因素疾病,特征是泪膜稳态的丧失并伴有眼表症状,其病因包括泪膜不稳定、泪液高渗性、眼表炎症与损伤和神经感觉异常[1]。D E作为眼科的常见疾病,血源性滴眼液的治疗方法的方式已经成为趋势。但由于多种因素的限制,不同专家或不同地区对血源性滴眼液的来源、制备、储存等存在不同的见解。本文对多种来源的血源性滴眼液进行阐述,总结了其制备方法、储存及安全性达成的共识,旨在通过更多的临床指南为患者提供个体化、高质量及有效的治疗策略。

1 血源性滴眼液

眼表是眼部一种特殊的组织结构,包括从上下眼睑皮肤和黏膜移行处到上下睑、球结膜返折处再到角巩膜缘和角膜的所有组织[2]。20世纪70年代,人们首次发现自体血清可用于治疗眼部化学性烧伤[3],而后沿用至治疗干燥综合征相关性DE患者[4],并逐渐在眼科治疗中流行,并且有研究总结这些血液制品的来源包括自体和供体,而供体来源又包括同种异体外周血血清和成人脐带血,并以血清、富血小板血浆(platelet-rich plasma,PRP)、富生长因子血浆(grow factor-rich plasma,PRGF)和血小板裂解液(platelet lysate,PL)的形式制备[5],近几十年来,血源性滴眼液已被广泛应用于各种眼表疾病的治疗,主要包括DE、持续性角膜上皮缺损(persistent epithelial defect,PED)、无菌性角膜溃疡、眼表烧伤、复发性角膜侵蚀(recurrent corneal erosion,RCE)、眼部移植物抗宿主病(ocular graft-versus-host disease,oGVHD)和角膜缘干细胞缺乏症(limbal stem-cell deficiency,LSTD)[6]。血源性产品未来可根据每个患者和眼表疾病的不同选择合适的生长因子及细胞因子含量,从而引入特制的血源性滴眼液(serum-derived eye drops,SED)[6],它是通过在眼表提供生长因子和细胞因子的活性混合物来刺激细胞增殖和迁移,从而模仿天然泪液所发挥的功能[6]
SED是包含碳水化合物、脂质、各种电解质、比泪液总浓度高十倍的蛋白质,以及凝血过程中血小板α颗粒脱颗粒释放的上皮营养生长因子的一种混合物[7-8]。由于这些特性,SED才被看作是一种有效且不良反应少的治疗方法。不仅如此,SED还是英国唯一一种营养泪膜替代品,具有生物学特性,可帮助提供促进上皮细胞更新和恢复稳态的眼表环境[9]

2 自体血清滴眼液

自体血清滴眼液(autologous serum eye drop,auto-SED)顾名思义,是由自体血清而制成的滴眼液,而这种滴眼液比传统疗法有潜在的优势,因为它不仅可以作为泪腺的替代品提供润滑,而且包含其他生化成分,使其更接近天然泪液[10]。有研究[11]结果表明:auto-SED被认为是安全、有效且可用于治疗各种眼表疾病的方法,包括原发性和继发性干燥综合征(Sj?gren syndrome,SS)引起的DE。

2.1 制备

血清是全血凝固后残留的成分,它包含存在于泪液中的许多成分。自体血清是从患者自身的外周血清中获得的。生产方法需考虑凝血时间、离心参数、稀释、储存时间和温度等因素,这些因素决定了最终产品的质量和特点。由于制备过程需要考虑的这些因素过于繁杂以及精细,迄今为止,尚未就制备auto-SED的技术细节达成共识。因此,用于治疗DE的SED的配方在很大程度上取决于国家或地区血液机构建立的独立血液中心的经验,以下是与生物医学卓越安全输血(biomedical excellence for safer transfusion,BEST)合作进行的一项最新调查发现的制备SED的方法[12]。简单地说,从患者身上取50~100 mL的全血,室温静置2 h。然后,以3 000 g离心15 min将血清与固体成分完全分离。接下来,将平衡盐溶液(balanced salt solution,BSS)或等渗液加入到上清液即血清中,直到达到所需的浓度,最终即制备完成。自体血清必须在?20 ℃下储存且最多储存3个月,它必须避光保存,以避免维生素A的退化[13]

2.2 稀释

BEST调查报告[14]显示:近一半的血液中心配药前稀释血清目的是降低潜在的抗增殖因子的生理浓度水平,如转化生长因子β(transforming growth factor β,TGF-β)。事实上,这项调查已经表明当角膜损伤后,在愈合时,TGF-β在肌成纤维细胞的成熟过程中起主要作用,它产生杂乱无章的细胞外基质,且该基质会导致透明度的损失。已知TGF-β具有剂量依赖性的抗增殖特性,其平均水平通常比天然泪液高5倍,所以通常将其稀释20%使用,避免带来影响上皮损伤愈合的潜在危害[15]。然而,血清的稀释会导致营养因子的水平下降,这也潜在地限制了它的疗效。等渗性滴眼液在眼表的应用能避免由于渗透压的波动引起眼表损伤的发生,这是一个特别的发现,即高渗透压(>308 mOsm/L)在重度DE的病理生理学中,被认为是关键的致病因素以及DE恶性循环的致病因素[16-17]。有了上述证据,就不能排除可能根据所治疗的疾病或其程度调整制剂的情况。

2.3 储存

血清制剂的建议最长保存时间存在显著的差异,部分是由于国际法律法规的差异。冷冻保存血清瓶以保持上皮营养因子的活性尤为重要,目前有研究表明血清制品可在-20 ℃保存6个月[18]

2.4 安全性

应避免接触受污染的auto-SED,尤其是DE患者,原因是这些患者由于存在眼表的病变而具有更高的感染风险。SED通常在开放式系统中制备,但在制备过程中仍然有潜在的污染风险。文献[19]已经报道了与污染有关的不良反应。
为提高安全性,市场上已经提出了密封的制备系统(将滴眼液包装进小瓶中或长管中,然后将其热封以生产一次性设备)[20-22],但是在许多血液中心,财务和物流障碍仍然难以克服。由于血清溶液环境有利于微生物的生长,在制备过程中必须谨慎,以确保滴眼液是在无菌状态下制备,且必须遵循适当的保存和使用说明[23]
auto-SED的其他缺点是经常抽血不方便,因为有些人怕静脉穿刺且治疗时间长。综上所述,auto-SED虽然在原理上可以作为一种有效的DE治疗方法,但不论是制备、稀释、储存还是安全性方面都有许多需要特别注意的因素,对自体血清治疗眼表损伤进行探索仍充满挑战。因为血清不是传统的血液产品,关于应将SED视为血液产品还是一种药物仍然存在争论[24]

3 同种异体血清滴眼液

同种异体血清滴眼液(allogeneic serum eye drop,allo-SED)就是使用供体来源制备的SED,据报道,S S和oGVHD代表2种可导致严重D E最常见的原因,这些患者的血清中可能含有高浓度的促炎性介质,对眼表具有潜在的伤害[25]。这种SED使用的适应证包括:患者自体血清不适合、无法使用或无法进行重复采血,包括病毒感染、败血症、严重贫血患者和多系统疾病的老年患者[26]。因此,使用allo-SED时我们要确认患者是否可用、有无禁忌证。另外,当滴眼液来自供体时,必须执行ABO血型匹配的程序,以确保供体和受体之间血型匹配。有一种选择是从AB血型献血者处获得,以保持单一的血型来源[27]
allo-SED的制备方法与auto-SED相同。血液被采集并室温储存到凝固。然后,将血清以3 000 g离心15 min,过滤,稀释至20%浓度[28]。因此,从健康供体获得的allo-SED已被引入作为一种可能的替代方案。

4 同种异体脐带血血清滴眼液

相对于allo-SED,脐带血血清(umbilical cord blood serum,UCBS)也有使用的适应证,它代表了针对全身性疾病或其他由于自身血清中存在炎症介质而可能禁止使用自体血清滴眼液的患者的可能疗法。它的优势之一在于可以从脐静脉收集大量血液,因此可以同时为多位患者准备[29-30]。与其他血液来源的制剂相比,UCBS的生长因子(grow factor,GF)浓度较高,例如表皮细胞生长因子(epidermal growth factor,EGF)、TGF-β、神经生长因子(nerve grow factor,NGF)和血管内皮生长因子(vascular endothelial growth factor,VEGF)[31]。另外由于UCBS含有抗菌成分如免疫球蛋白G、溶菌酶和补体,因此具有抑菌效果[32]。研究[33]表明:从年轻母亲(<30岁)采集的血液样本具有较高的EGF浓度,分娩时间越长(>6 h),CD34+细胞含量越高,这种预选择可能有助于在EGF中获得理想浓度的UCBS,无需达到GF实验室剂量即可治愈角膜伤口。每一种GF都有选择性地调节角膜愈合过程中涉及的细胞过程,因此可根据角膜病变的类型和严重程度,针对性地提供特定的GF[34]
在顺产和剖宫产时收集母亲的脐带血血清。从分娩后的脐静脉收集10 ~ 20 mL的脐带血。血液在室温下凝固2 h,3 000 g离心15 min后,在无菌条件下,在层流罩中仔细分离血清。然后,用BSS稀释达到20%的浓度。将血清注入无菌的1 mL小瓶中,用紫外线灯照射保护。
为避免血源性疾病的传播,必须配制前必须进行HCV(丙型肝炎病毒)、HBV(乙型肝炎病毒)、HIV(人类免疫缺陷病毒)、梅毒、弓形虫和CMV(巨细胞病毒)的检测。可见,来源于USBS的SED也可作为一种DE的治疗方法。

5 血小板源性滴眼液

血小板在凝血过程中发挥重要作用,在伤口愈合过程中起主要作用,因为它们的α颗粒中含有高浓度的GF和凝血因子,例如血小板来源生长因子(platelet derived growth factor,PDGF)、TGF-β、EGF[35]。最近已经引入了各种制剂(即由来源于PRP、PRGF和血小板裂解产物的制剂)作为不同眼表疾病的可能疗法。在血小板源滴眼液中,自体来源的血液产品仍然为DE患者使用最多的,而针对其他应用领域开发的作为滴眼液治疗的同种异体PRP仍处于起步阶段。

5.1 PRP

富血小板血浆是从血小板浓度超过1×106/mL的血浆中获得的。第1次低力离心(200~600 g,10 min)将全血分成三层,将上层和浅层表皮层转移到另一个无菌试管中,以更高的速度进行第二个离心步骤(700~2 300 g,10 min),以浓缩血小板。将上2/3的体积丢弃,而将下1/3匀浆,通过轻轻摇动试管以产生PRP。该产品可以未经稀释或在BSS中稀释使用,密封在小瓶中,在4 ℃最多保存1周,而在20℃可保存更长时间[36]

5.2 PRGF

在Endoret系统离心机中,将收集的血液在室温下以580 g离心8 min。离心后,使用Endoret眼科试剂盒收集PRGF,避开含有白细胞的层次。PRGF滴眼液在37 ℃下孵育1 h;将获得的上清液在56 ℃下热处理60 min,以消除补体和其他免疫学成分,并获得所谓的“免疫安全PRGF”。最后,将血浆上清液过滤,等分并保存在?20 ℃。PRGF滴眼液在?20 ℃保质期为3个月[37]。在一项研究[38]中,所有分析的GF水平在每次不同储存条件下都保持不变。在任何研究时间或温度下,使用PRGF滴眼液后,均未观察到角化细胞增殖活性的差异。最后,PRGF滴眼液中没有微生物污染。

5.3 血小板裂解产物

从每位患者中收集60 mL抗凝的外周血,然后离心以获得自体血小板浓缩物。将浓缩液冷冻并融化以裂解血小板并释放GF。然后将裂解物用无菌平衡盐溶液稀释,并分装成30份备用的无菌剂量。在制备时会采集用于微生物学研究的样品。最终应在?20 ℃冰箱中冷冻存放,最长保存45 d[39-40]
通过上述研究,我们可以了解到血小板源性滴眼液有多种形式,其制备方法、储存方式等不尽相同,使用者们可以根据自己的需求以及适应证来选择合适的滴眼液。

6 结语

SED能提供活性物质比如谷胱甘肽和细胞因子等眼泪中的活性成分,模拟泪液的功能,可作为治疗DE等眼表疾病的一种新兴治疗选择。目前多项指南皆建议SED对急性和慢性严重眼表疾病的患者(包括严重DE患者、持续性和复发性角膜上皮缺损患者、神经营养性角膜病变患者)以及需要手术支持治疗的患者治疗有所帮助。现如今,自体来源的血清制品相较于异体来源使用更广且技术更成熟,但异体来源的产品正在逐渐进步。对于SED的应用前的制备参数、保存条件、临床治疗策略等的选择和评估尤为重要。目前,我们仍需更多的随机临床试验来为国际公认的统一指南标准提供更多的证据,从而确定血清成分中的生物物种可变性以提高最终的血清眼药水的质量,进一步确定检测有效性的生物标志物,以及确定每种适应症SED治疗的最佳频率、计量和持续时间,以确定治疗的有效性,使得血液疗法能够广泛而成熟地运用于眼科疾病的治疗。
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1、张梦婷. 养阴清中汤治疗阴虚湿热型干眼症的临床疗效评价[D].南京中医药大学,2023.
2、何婷. 滋阴生津法治疗白涩症肝肾阴虚型疗效观察[D].云南中医药大学,2023.
3、亚洲干眼协会中国分会,海峡两岸医药卫生交流协会眼科学专业委员会眼表与泪液病学组,中国医师协会眼科医师分会眼表与干眼学组.中国干眼专家共识:免疫性疾病相关性干眼(2021年)[J].中华眼科杂志,2021,57(12):898-907.
4、何继武,段鹏,叶琳等.超说明书用药的医学伦理分析——以眼科为例[J].医学与哲学,2021,42(24):17-20.
1、国家自然科学基金 (81660158);江西省自然科学基金 (2016ACB21017);江西省青年科学基金 (20151BAB215016, 20161BAB215198);江西省新型冠状病毒感染肺炎疫情应急科技攻关项目 (202011-2);江西省重点研发项目 (20151BBG70223, 20181BBG70004);江西省杰出青年人才计划 (S2019RCQNB0259)。
This work was supported by the National Natural Science Foundation (81660158), Natural Science Foundation of Jiangxi Province (2016ACB21017), Youth Science Foundation of Jiangxi Province (20151BAB215016, 20161BAB215198), Emergency Science and Technology Project for COVID-19 of Jiangxi Province (20201102), Key Research Foundation of Jiangxi Province (20151BBG70223, 20181BBG70004), and Excellent Talents Development Project of Jiangxi Province (S2019RCQNB0259), China.()
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