Alpha-Gal antigen and immunity risk control of animal-derived medical devices

doi:10.3969/j.issn.2095-4344.2014.25.020

Abstract

Abstract:

BACKGROUND: Medical devices from animals are commonly used in clinical application. Despite their efficiency is widely accepted, their safety, especially immunity has been concerned.
OBJECTIVE: To investigate immunity risk control to medical devices from animals for safety consideration.
METHODS: Using “α-Gal antigen, immunity, xenotransplantation” in Chinese and English as the key words, the first author conducted a computer search of Science direct database (www.sciencedirect.com), Wiley-Blackewell database (http://onlinelibrary.wiley.com) and Wanfang database (www.wanfang.com.cn) through screening the titles and abstracts.
RESULTS AND CONCLUSION: Increasing evidence shows that, Gal α1-3Gal antigen (α-Gal antigen) is recognized as the major antigen and abundantly expressed on glycoconjugates of non-primate mammals and New World monkeys. In contrast, the α-gal epitope is not expressed on glycoconjugates of humans and Old World monkeys. Instead, they produce a very large amount of natural anti-α-Gal antibody that specifically binds the α-gal epitope. The binding of human natural anti-α-Gal to α-gal epitopes expressed on non-primate mammal animals was expected to be unique immunological barrier in xenotransplantation. Therefore, it is important to choose raw materials, reduce or eliminate the α-Ggal epitope, establish highα-Ggal epitope detection methods with high sensitivity and good repeatability for achieving a greater safety and efficiency of medical devices from animals.

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程

全文链接：

Key words: antigen, transplantation, heterologous, review

CLC Number:

R318

Ke Lin-nan, Fang Yu, Shan Yong-qiang, Feng Xiao-ming, Xu Li-ming, Wang Chun-ren. Alpha-Gal antigen and immunity risk control of animal-derived medical devices[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(25): 4051-4056.

Figures/Tables 1

2.1 α-Gal抗原的生物化学 2.1.1 α-Gal抗原 α-Gal抗原本质上就是一组糖蛋白或糖脂类物质。研究者利用气-质联用色谱、核磁、质谱等技术对这些糖基化合物的结构进行了研究和表征，发现它们在分子结构上具有相同的特征，即糖链末端含有Galα1-3Galβ1-4GlcNAc结构[9-11]。从哺乳动物组织中分离出的很多糖脂，如鞘糖脂上都发现了α-Gal抗原的存在[12-14]。糖蛋白如甲状腺球蛋白、纤维蛋白、免疫球蛋白及层粘连蛋白的糖链末端也都含有α-Gal抗原[15-18]。 α-Gal抗原的合成依赖于1，3半乳糖转移酶[UDP-Gal: -galactosyl 1-3-galactosyltransferase，简称 1,3GT]和iGb3合成酶的催化，尿苷二磷酸半乳糖(UDP-Gal)作为半乳糖供体，在α1，3GT的催化下，将半乳糖残基以α1-3键连接至到N-乙酰基乳糖胺(Gal β1-4 GlcNAc-R)的末端，最终形成了α-Gal抗原[19]，其反应可简单表示为：Gal β1-4 GlcNAc-R+UDP-Gal Galα1-3 Gal β1-4 GlcNAc-R+UDP。在非灵长类哺乳动物及新世纪猴的细胞及组织里，α1,3GT是有活性的，而在旧世纪猴及人类体内，α1,3GT的基因在进化过程中发生了移码突变，导致α1，3GT失活，无法催化形成α-Gal抗原，所以在人类及旧世纪猴体内不表达α-Gal抗原[20-23]。 1984年，Galili等[24]首次发现在正常人体血清中存在能与α-连接半乳糖特异结合的抗体，即抗α-Gal抗体，这种抗体的含量大约占人血清中IgG总量的1%。在胃肠道微生物抗原的刺激下，人体终生都会产生抗α-Gal抗体[25]。Galili的研究团队对抗α-Gal抗体与α-Gal抗原结合位置进行了深入研究[26]，他们从兔血红细胞中分离出一系列结构相似的鞘糖脂，用免疫印染法考察抗α-Gal抗体对它们的结合情况，并采用已知结构的寡糖进行了血凝抑制试验。试验结果表明：尽管糖基顺序和端基差向异构一致，抗α-Gal抗体也可区分出糖基末端半乳糖残基的连接位置，抗α-Gal抗体只与末端含有Gal α1-3 Gal连接的糖基结合，不与末端含有Gal α1-4 Gal的糖基结合。 2.1.2 α-Gal抗原在动物中的表达及分布 α-Gal抗原在大鼠[9，34]、兔[9, 11，16]、羊[9，11]、牛[9-10，17]、猪[9，11，18，27-33]，及小鼠等动物的组织及红细胞中均有表达[35-37]。在不同动物、不同组织中α-Gal抗原分布不同，表达强弱不同。以猪为例，α-Gal抗原广泛分布于猪的各种组织中[30-33]，在猪体内的所有血管内皮细胞α-Gal抗原都有表达；猪的毛细血管、小动脉和小静脉等小血管内皮细胞中都有大量α-Gal抗原表达，而在主动脉内皮细胞中只有少量表达，心脏的毛细血管中有高浓度表达，而心肌细胞无此抗原，肝脏和肾脏的实质中表达大量α-Gal抗原。肾脏中α-Gal抗原的表达呈差异性分布，近曲小管表达最强，远曲小管只有中等程度的表达，肾小球和集合管中则无表达。肺脏的肺泡内壁和支气管血管一样表达很强。成年猪的胰腺组织除血管和管内膜表达外，其他部位，如胰岛细胞均无此抗原分布。猪的骨、软骨及角膜基质中均有表达。小鼠和猪的α-Gal抗原分布存在显著不同，在小鼠内皮细胞中虽有α-Gal抗原的表达，却没有猪的密集[38]。 2.2 α-Gal抗原与动物源性医疗器械免疫原性风险目前已有很多商品化的动物源性医疗器械产品，如生物瓣、组织补片、骨修复材料、骨填充材料，人工皮肤、异种脱细胞真皮基质及可吸收缝线等[39-41]。钙化是生物瓣在临床使用上面临的最大问题，可能导致生物瓣的降解，缩短其使用寿命[42-44]，特别是对年轻的患者或儿童，这种风险更为突出[45-48]。引起生物瓣钙化、降解的原因有很多，免疫反应是其中重要的因素之一[49-53]。人体在接受了生物瓣植入手术后，α-Gal抗原作为主要的抗原，会引发针对抗α-Gal的炎症反应[54]。Kasimir等[55]在已上市的猪生物瓣中检测到α-Gal抗原的存在。Park等[56]在接受了生物瓣植入手术的儿童血清中检测出了抗α-Gal抗体，包括IgM和IgG两种亚型，这两种亚型的滴度会随着时间发生变化。Mangold等[57]和Konakci等[58]也得到了相似的研究结果，在植入生物瓣后的一段时间，人体内抗α-Gal抗体增加，并伴有α-Gal特有的体液免疫应答。Stone等[59]发现猪、牛的软骨中均有少量α-Gal抗原表达，分别将猪、牛的软骨植入猕猴来考察动物软骨作为异源材料植入人体的可能性。在2个月的评价期内，所有猕猴都显示出了强烈的体液免疫应答，组织学观察证明了慢性排斥反应的发生。对脱细胞小肠黏膜下层植入人体的研究发现，患者在植入小肠黏膜下层后，出现了抗α-Gal抗原的抗体反应，但是没有出现异种植入物临床排斥反应和最小的术后并发症[60]。 2.3 降低由α-Gal抗原引起免疫排斥反应的方法目前降低由α-Gal抗原引起免疫排斥反应的策略主要有以下两方面：一是从受者入手，二是从供体入手，前者主要考虑如何减少受者体内抗体的滴度或特异性，后者则主要考虑如何减少供体的α-Gal抗原数量或降低α-Gal抗原的活性[61]。对于动物源性医疗器械产品而言，后者则是更常用且更为重要的。目前主要有以下方法： 2.3.1 去除抗体利用血浆分离置换仪与Gal亲合层析柱用体外免疫吸附的方法可以去除抗α-Gal的抗体，但是一段时间后，抗体量会逐渐恢复，恢复的速度依赖于免疫抑制剂的使用。另外用合成的可溶性α-Gal低聚糖也可以去除血浆中抗α-Gal抗原的抗体，但是α-Gal低聚糖拮抗抗α-Gal抗原的抗体这种方法只能延迟，而不能完全阻止排斥反应的发生[62]。 2.3.2 降低α-Gal抗原的表达酶消化法去除α-Gal抗原：α-半乳糖苷酶可以作用于Gal α1-3Gal β1-4GlcNAc-R结构中2个半乳糖之间的α1,3糖苷键，将最外端的半乳糖水解，从而降低或去除细胞表面的α-Gal抗原[63]。Cairns等[64]利用α-半乳糖苷酶处理猪内皮细胞和淋巴细胞后，再用与半乳糖残基高度亲和的植物凝集素Gariffonia Simplicifolia IB4(GSI-B4)检测细胞内的α-Gal抗原，发现经过α-半乳糖苷酶处理后，α-Gal抗原数量显著下降。LaVecchio等[65]发现绿咖啡豆α-半乳糖苷酶处理后的猪内皮细胞，与人和猕猴血清的反应力降低了59%到90%，而且细胞毒性也伴随着下降。人重组α-半乳糖苷酶和咖啡豆α-半乳糖苷酶对去除α-Gal抗原有同样的作用[57]。Stone等[66]将猪的韧带经重组α-半乳糖苷酶处理后，移植入6例前交叉韧带损伤患者，2年随访结果表明：在6例移植患者中有5例成功，并且通过了所有的功能稳定性评估。半乳糖苷酶去除α-Gal抗原只是暂时的，因为细胞内α1,3GT仍然存在，它能重新合成α-Gal抗原。酶竞争法去除α-Gal抗原：利用某些糖基转移酶可以与α1,3GT竞争N-乙酰乳糖胺基底物，从而减少α-Gal抗原的表达。人体内存在的α1,2-岩藻糖转移酶(α1,2- fucosyltranserase，α1,2FUT)和唾液酸糖基转移酶(包括α2,3-Sialytransferases, α2,3ST和α2,6- Sialytransferases, α2,6 ST)，与α1,3GT功能相似，都以N-乙酰乳糖胺基为底物，分别将岩藻糖基和唾液酸转移至该底物，从而产生岩藻糖基化的N-乙酰半乳糖胺(H抗原)和N-唾液酸化的乙酰基乳糖胺。在非灵长类哺乳动物体内，岩藻糖基化的N-乙酰半乳糖胺不能再受α1,3GT的催化接受半乳糖基残基。通过转基因的方法将α1,2FUT[67-69]或α2,3ST[70]引入猪的细胞中与α1,3GT形成竞争，用岩藻糖或唾液酸替代半乳糖，诱导竞争抑制物高表达，从而抑制α-Gal抗原的表达。将α2,3ST基因引入猪的内皮细胞中，α2,3ST基因的过表达使猪内皮细胞对人天然抗体的抗原性下降77%，并且α-Gal抗原数量下降并伴有α1,3GT活力的下降[71]。有研究表明，将α1,2FUT基因和α-半乳糖苷酶基因联合转入猪胚胎成纤细胞中，可以发挥两种酶协同作用，既可直接降低抗原数量，又可通过增加H抗原的表达减少α-Gal抗原的产生[72]。以上方法尽管可以减少移植物体内的α-Gal抗原的表达，但是不能完全避免HAR的产生，因为在移植物中极少量的α-Gal抗原，如每个细胞中低至1 105个α-Gal抗原，都会引发抗α-Gal抗体的产生，从而引起强烈的免疫反应[73]。基因敲除α1,3GT：生产不表达α-Gal抗原的动物为克服或消除异种移植排斥反应提供了新思路。研究者利用同源基因重组的方法得到αGT基因缺失的活细胞克隆，然后通过核转移技术成功克隆出αGT基因缺失的猪，并证实了这一方法的可行性[74-76]。但是基因敲除猪的存活率低一直没有解决。 2.4 α-Gal抗原检验方法 2.4.1 免疫组织化学法目前对α-Gal抗原检测报道最多的是免疫组织化学法或免疫细胞化学技术，即通过标记抗体、凝集素的显色剂(荧光素、酶、同位素等)显色来确定组织细胞内α-Gal抗原的分布及数量。如西非单叶豆凝集素(简称GS-IB4)可以与末端带有α-半乳糖结构的糖基化合物结合[77]。将GS-IB4与荧光物质如异硫氰酸酯(FITC)偶联后，作用于组织或细胞，经过激发产生荧光，再用荧光显微镜观察或流式细胞仪可以对α-Gal抗原定位及定量检测[78]。由于GS-IB4可以与α-半乳糖特异性结合，对于非Gal α1-3Gal连接的半乳糖也有亲合力，因此对于α-Gal抗原的识别，GS-IB4并非完全特异性的[54]。另外该方法灵敏度低，对于α-Gal抗原数低于5×104/每个细胞，将很难检测到，无法客观地反映组织中α-Gal抗原的多少[78]。Galili等[78]应用杂交瘤技术免疫小鼠制备了抗α-Gal的单克隆抗体(简称M86)，它是一种IgM抗体，对α-Gal抗原的亲和力强于GS-IB4[79]。这种抗体可以与α-Gal抗原特异性结合，再用酶标二抗与之反应，形成抗原-抗体-酶标二抗复合物，加底物显色，根据底物显色反应检测α-Gal抗原在动物组织中的分布、定量[80-81]。 2.4.2 竞争酶联免疫吸附法(ELISA)定量检测 Galili 等[78]建立了ELISA竞争法定量检测α-Gal抗原的方法。该方法以Gal α1-3-BSA作为包被抗原包被酶标板，样品中加入过量的M86抗体充分反应后，通过高速离心将未被结合的M86抗体与α-Gal抗原-M86抗体结合物分离，未被结合的M86抗体与酶标板上包被的Gal α1-3-BSA结合，然后加入酶标记的第二抗体，最后加入酶底物显色。通过与已知α-Gal抗原数的兔血红细胞所建立的抑制曲线比较，可以得到样品中α-Gal抗原数。Galili用建立的ELISA法对不同动物的细胞，包括基因敲除小鼠的脾细胞及小鼠不同器官上的α-Gal抗原数进行了测定。由于此方法用的是兔血红细胞作为对照，而兔血红细胞上α-Gal抗原数为估算值，所以该方法只能作为α-Gal抗原数半定量方法。Naso等[79]在该方法的基础上，针对生物组织建立了α-Gal抗原定量ELISA检测方法，并对脱细胞处理工艺前后猪心脏瓣中α-Gal抗原数进行测定。"

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