在α-1抗胰蛋白酶存在的情况下培养不纯的人胰岛组分可防止胰岛素裂解并改善胰岛回收率。
Culture of impure human islet fractions in the presence of alpha-1 antitrypsin prevents insulin cleavage and improves islet recovery.
作者信息
Loganathan G, Dawra R K, Pugazhenthi S, Wiseman A C, Sanders M A, Saluja A K, Sutherland D E R, Hering B J, Balamurugan A N
机构信息
Schulze Diabetes Institute, Department of Surgery, University of Minnesota, Minneapolis, Minnesota 55455, USA.
出版信息
Transplant Proc. 2010 Jul-Aug;42(6):2055-7. doi: 10.1016/j.transproceed.2010.05.119.
BACKGROUND
Exocrine tissue is commonly cotransplanted with islets in autografting and allotransplantation of impure preparations. Proteases and insulin are released by acinar cells and islets, respectively, during pretransplantation culture and also systemically after transplantation. We hypothesized that released proteases could cleave insulin molecules and that addition of alpha-1 antitrypsin (A1AT) to impure islet cultures would block this cleavage, improving islet recovery and function.
METHODS
Trypsin, chymotrypsin, and elastase (TCE) activity and insulin levels were measured in culture supernates of pure (n = 5) and impure (n = 5) islet fractions, which were isolated from deceased donors. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to detect insulin after incubation with proteases. We assessed the effects of A1AT supplementation (0.5 mg/mL; n = 4] on TCE activity, insulin levels, culture recovery, and islet quality. The ultrastructure of islets exposed to TCE versus control medium was examined using electron microscopy (EM).
RESULTS
Protease (TCE) activity in culture supernatants was indirectly proportional to the percentage purity of islets: pure, impure, or highly impure. Increasingly lower levels of insulin were detected in culture supernatants when higher protease activity levels were present. Insulin levels measured from supernatants of impure and highly impure islet preparations were 61 +/- 23.7% and 34 +/- 33% of that in pure preparations, respectively. Incubation with commercially available proteases (TCE) or exocrine acinar cell supernatant cleaved insulin molecules as assessed using SDS-PAGE. Addition of A1AT to impure islet preparations reduced protease activity and restored normal insulin levels as detected using enzyme-linked immunosorbent assay (ELISA) and SDS-PAGE of culture supernates. A1AT improved insulin levels to 98% +/- 1.3% in impure and 78% +/- 34.2% in highly impure fractions compared with pure islet fractions. A1AT supplementation improved postculture recovery of islets in impure preparations compared with nontreated controls (72% +/- 9% vs 47% +/- 15%). Islet viability as measured using membrane integrity assays was similar in both the control (98% +/- 2%) and the A1AT-treated groups (99% +/- 1%). EM results revealed a reduction or absence of secretory granules after exposure to proteases (TCE).
CONCLUSION
Culture of impure human islet fractions in the presence of A1AT prevented insulin cleavage and improved islet recovery. A1AT supplementation of islet culture media, therefore, may increase the proportion of human islet products that meet release criteria for transplantation.
背景
在自体移植和异体移植不纯胰岛制剂时，外分泌组织通常与胰岛一起进行共移植。在移植前培养期间，腺泡细胞和胰岛分别释放蛋白酶和胰岛素，移植后全身也会释放。我们推测，释放的蛋白酶可能会切割胰岛素分子，并且在不纯的胰岛培养物中添加α-1抗胰蛋白酶（A1AT）会阻止这种切割，从而改善胰岛的恢复和功能。
方法
测量从已故供体分离的纯（n = 5）和不纯（n = 5）胰岛组分培养上清液中的胰蛋白酶、糜蛋白酶和弹性蛋白酶（TCE）活性以及胰岛素水平。用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）检测与蛋白酶孵育后的胰岛素。我们评估了添加A1AT（0.5 mg/mL；n = 4）对TCE活性、胰岛素水平、培养物恢复和胰岛质量的影响。使用电子显微镜（EM）检查暴露于TCE的胰岛与对照培养基的超微结构。
结果
培养上清液中的蛋白酶（TCE）活性与胰岛的纯度百分比成反比：纯的、不纯的或高度不纯的。当蛋白酶活性水平较高时，在培养上清液中检测到的胰岛素水平越来越低。不纯和高度不纯胰岛制剂上清液中测得的胰岛素水平分别为纯制剂的61±23.7%和34±33%。使用SDS-PAGE评估，与市售蛋白酶（TCE）或外分泌腺泡细胞上清液孵育可切割胰岛素分子。向不纯的胰岛制剂中添加A1AT可降低蛋白酶活性，并通过酶联免疫吸附测定（ELISA）和培养上清液的SDS-PAGE检测恢复正常胰岛素水平。与纯胰岛组分相比，A1AT将不纯组分中的胰岛素水平提高到98%±1.3%，将高度不纯组分中的胰岛素水平提高到78%±34.2%。与未处理的对照相比，添加A1AT可改善不纯制剂中胰岛培养后的恢复（72%±9%对vs 47%±15%）。使用膜完整性测定法测量的胰岛活力在对照组（98%±2%）和A1AT处理组（99%±1%）中相似。EM结果显示，暴露于蛋白酶（TCE）后分泌颗粒减少或消失。
结论
在A1AT存在的情况下培养不纯的人胰岛组分可防止胰岛素切割并改善胰岛恢复。因此，在胰岛培养基中添加A1AT可能会增加符合移植释放标准的人胰岛产品的比例。
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