Recombinant Nidogen-1 Significantly Improves Survival of Hypoxic Human Islets

D. Brandhorst
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引用次数: 1

Abstract

Daniel Brandhorst,1,2 Heide Brandhorst,1,2 Samuel Acreman,1,2 Yukari Kimura,1,2 Shannon Layland,3 Katja Schenke-Layland,3 Paul R.V. Johnson1,2 1 Research Group for Islet Transplantation, Nuffield Department of Surgical Sciences, John Radcliffe Hospital, University of Oxford, Oxford, United Kingdom 2 Oxford Consortium for Islet Transplantation, Oxford Centre for Diabetes, Endocrinology, and Metabolism (OCDEM), Churchill Hospital, University of Oxford, Oxford, United Kingdom 3 Natural and Medical Sciences Institute, Eberhard Karls University, Tübingen, Germany Aim Islet isolation essentially requires dissociation of the islet basement membrane by collagenolytic enzymes. Basement membrane loss is associated with reduced islet function and viability. Previous studies demonstrated that individual extracellular matrix proteins (ECMPs) can increase islet survival pre- and post-transplant. In the present study, we tested our hypothesis, that the combination of different ECMPs, particularly those forming suprastructures, are more efficient than individual ECMPs to protect human islets from hypoxia-induced damage. In contrast to previous studies, we dissolved ECMPs in the media rather than to coat culture surfaces. Methods Islets, isolated from pancreases of 11 human DBD donors (50±2 years, 29.3±1.2 BMI, 5.7±0.3 hours CIT), were cultured for 3–4 days in 2% oxygen and suspended in CMRL 1066 (2% FCS) supplemented with either 40 µg/mL of dissolved collagen-IV, 10 µg/mL laminin-521 or 12.5 µg/mL nidogen-1 used individually or as combination. Sham-treated islets (STIs) cultured without ECMPs served as controls. Post-culture characterisation included IEQ yield or islet number (IN), viability (FDA-PI), early plus late apoptosis (annexin V-PI), glucose stimulation index (SI: 2 vs 20 mM) and reactive oxygen species production. Parameters were normalised to IEQ, related to pre-culture data if appropriate and presented as mean ± SEM. Statistical analysis was performed by Friedman test followed by Dunn’s multiple comparison. Results Compared with STI (41±7%), post-culture recovery was higher when hypoxic islets were treated with collagen-IV (64±7%, p<0.001), laminin-521 (57±6%, p<0.01) or nidogen-1 (65±6%, p<0.001) used individually or combined (61±7% p<0.001). This correlated with islet fragmentation (IN/IEQ ratio) that was lower when collagen-IV (116±13%, p<0.001), laminin-521 (114±12%, p<0.01), nidogen-1 (121±12%, p<0.01) or combined ECMPs (119±13%, p<0.001) were compared with STIs (155±16%). Reactive oxygen species production in STIs was substantially reduced by 71±6% (NS), 73±6% (p<0.05), 90±2% (p<0.001), and 87±4% (p<0.001) in presence of collagen-IV, laminin-521, nidogen-1 or combined ECMPs, respectively. This resulted in improved viability (83±7% [p<0.01], 79±9% [p<0.01], 84±7% [p<0.001], 83±8% [p<0.001]) compared with STIs (63±7%). While individual ECMPs stabilised or reduced pre-culture apoptosis (94±17% [p<0.05], 117±16% [p<0.05], 68±13% [p<0.001]), combined ECMPs (171±18%, NS) were equal to STIs (196±28%). STIs did not adequately secrete insulin after glucose challenge (SI 0.97±0.13) in contrast to the physiological insulin response after treatment with collagen-IV (1.76±1.18 [p<0.01]), laminin-521 (1.53±0.25 [NS]), nidogen-1 (2.27±0.67 [p<0.01]) or combined ECMPs (1.95±0.25 [p<0.05]). Conclusion Among the three individual ECMPs tested, nidogen-1 appears to be most effective to protect human islets from hypoxia-induced damage. As its protective efficiency partially exceeds that of combined ECMPs, we have to reject our hypothesis. Further studies are required to clarify whether collagen-IV, laminin-521 and nidogen-1 spontaneously assemble to suprastructures in vitro.  
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重组Nidogen-1显著提高缺氧人胰岛的存活率
Daniel Brandhorst,1,2 Heide Brandhorst,1,2 Samuel Acreman,1,2 Yukari Kimura,1,2 Shannon Layland,3 Katja Schenke-Layland,3 Paul R.V. johnson 1,2 1,2英国牛津大学牛津大学约翰拉德克利夫医院纳菲尔德外科医学院胰岛移植研究组2牛津胰岛移植联盟,牛津大学糖尿病、内分泌和代谢中心(OCDEM),牛津大学丘吉尔医院,牛津,牛津,英国3 Eberhard Karls大学自然与医学研究所,德国宾根(tbingen) Aim胰岛分离本质上需要用胶原溶解酶解离胰岛基底膜。基底膜损失与胰岛功能和生存能力降低有关。先前的研究表明,单个细胞外基质蛋白(ECMPs)可以增加移植前和移植后胰岛的存活率。在本研究中,我们验证了我们的假设,即不同ecmp的组合,特别是那些形成上层结构的ecmp,比单个ecmp更有效地保护人类胰岛免受缺氧引起的损伤。与之前的研究相反,我们将ecmp溶解在培养基中,而不是包覆在培养表面。方法从11例DBD供体(50±2岁,29.3±1.2 BMI, 5.7±0.3小时CIT)胰腺中分离胰岛,在2%氧气条件下培养3-4天,悬浮在CMRL 1066 (2% FCS)中,分别添加40µg/mL溶解的collagen-IV、10µg/mL laminin-521或12.5µg/mL nidogen-1单独或联合使用。无ecmp培养的假处理胰岛(STIs)作为对照。培养后的特征包括IEQ产量或胰岛数量(IN)、活力(FDA-PI)、早期和晚期细胞凋亡(膜联蛋白V-PI)、葡萄糖刺激指数(SI: 2 vs 20 mM)和活性氧产生。参数归一化为IEQ,如果合适,与培养前数据相关,并以平均值±SEM表示。采用Friedman检验和Dunn多重比较进行统计分析。结果与STI(41±7%)相比,单独或联合使用collagen-IV(64±7%,p<0.001)、laminin-521(57±6%,p<0.01)或nidogen-1(65±6%,p<0.001)治疗缺氧胰岛的培养后恢复率更高(61±7%,p<0.001)。这与胰岛碎片化(IN/IEQ)相关,胶原- iv(116±13%,p<0.001)、层粘连蛋白-521(114±12%,p<0.01)、氮化原-1(121±12%,p<0.01)或联合ECMPs(119±13%,p<0.001)比sti(155±16%)更低。胶原- iv、laminin-521、nidogen-1或联合ECMPs存在时,sti中活性氧的产生分别显著减少71±6% (NS)、73±6% (p<0.05)、90±2% (p<0.001)和87±4% (p<0.001)。结果表明,与性传播感染(63±7%)相比,生存能力(83±7% [p<0.01], 79±9% [p<0.01], 84±7% [p<0.001], 83±8% [p<0.001])有所提高。单个ECMPs稳定或减少培养前细胞凋亡(94±17% [p<0.05], 117±16% [p<0.05], 68±13% [p<0.001]),联合ECMPs(171±18%,NS)与STIs(196±28%)相等。与使用胶原- iv(1.76±1.18 [p<0.01])、层粘连蛋白-521(1.53±0.25 [NS])、nidogen-1(2.27±0.67 [p<0.01])或联合ECMPs(1.95±0.25 [p<0.05])治疗后的生理性胰岛素应答相比,STIs在葡萄糖刺激后不能充分分泌胰岛素(SI 0.97±0.13)。结论在三种不同的ECMPs中,nidogen-1似乎对保护人类胰岛免受缺氧损伤最有效。由于其保护效率部分超过联合ecmp,我们不得不拒绝我们的假设。需要进一步的研究来阐明胶原- iv、层粘连蛋白-521和nidogen-1是否在体外自发组装到上层结构上。
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