A novel approach to xenotransplantation combining surface engineering and genetic modification of isolated adult porcine islets
A novel approach to xenotransplantation combining surface engineering and genetic modification of isolated adult porcine islets
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DOI：
10.1016/j.surg.2004.05.031
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发表时间：
2004-09-01
期刊：
影响因子：
3.8
通讯作者：
Eckhoff, DE
中科院分区：
文献类型：
--
作者：
Contreras, JL;Xie, D;Eckhoff, DE
背景。对异胰岛的有效细胞保护将规避胰岛移植（PIT）的主要组织限制。使用聚[乙二醇]（PEG）衍生物的细胞表面工程可以成功地阻止抗体与表面抗原结合。抗凋亡 Bcl-2 基因的基因转移已被证明可以降低异种反应性天然抗体和补体介导的细胞毒性。在这项研究中，我们评估了经过表面工程改造以过表达 Bcl-2 的猪胰岛的存活率和功能。方法。将 PEG 衍生物掺入胰岛表面，并在分离后 24 小时内完成腺病毒介导的 Bcl-2 (AdBcl-2) 基因转移。通过胰岛细胞内乳酸脱氢酶释放和使用膜完整性染色的显微分析来评估人异种反应性天然抗体诱导的细胞毒性：通过静态孵育和门静脉内输注（5000 IEQ）到用人淋巴细胞重建的糖尿病NOD-SLID小鼠（5 X 10（8）/腹膜内/PIT前15天）后评估胰岛功能。结果。在掺入 PEG-单琥珀酰亚胺基琥珀酸酯 (MSPEG) 或用白蛋白“末端”封端的 PEG-二琥珀酰亚胺基琥珀酸酯 (DSPEG) 后，无论有或没有 Bcl-2 基因转移，胰岛活力、形态和功能均没有显着变化。与对照组相比，用 MSPEG 处理的胰岛的乳酸脱氢酶释放显着减少（分别为 41.2 +/- 3 vs 72.1 +/- 7，P < .05）。 DSPEG 或 AdBcl-2 实现了进一步的保护。 DSPEG + AdBcl-2 实现了最大的细胞保护（15.5 +/- 4.9%，P < .001）。移植后 48 小时内，100% 接受对照胰岛的动物 (n = 6) 的非空腹血糖 >200 mg/dL。相比之下，在观察期间，接受 DSPEG + AdBcl-2 修饰胰岛的动物中 100% 达到了正常血糖。结论。功能化 PEG 衍生物的表面工程与 Bcl-2 基因修饰相结合，显着减少了 PIT 后的胰岛损失。这项新技术的应用可以改善异胰岛移植的结果。
Background. Effective cytoprotection to xenoislets would circumvent the major tissue limitation for pancreatic islet transplantation (PIT). Cell-surface engineering with poly [ethylene glycol] (PEG) derivatives can successfully prevent antibody binding to the surface antigens. Gene transfer of the antiapoptotic Bcl-2 gene has been shown to decrease cytotoxicity mediated by xenoreactive natural antibodies and complement. In this study, we assessed survival and function of surface-engineered porcine islets genetically modified to overexpress Bcl-2.Methods. Incorporation of PEG derivatives into the islet surface and adenovirus-mediated gene transfer of Bcl-2 (AdBcl-2) was accomplished within 24 hours post-isolation. Cytotoxicity induced by human xenoreactive natural antibodies was evaluated by islet intracellular lactate dehydrogenase release and microscopic analysis using membrane-integrity staining: Islet functionality was assessed by static incubation and after intraportal infusion (5000 IEQ) into diabetic NOD-SLID mice reconstituted with human lymphocytes (5 X 10(8)/intraperitoneally/15 days before PIT).Results. No significant change in islet viability, morphology, and functionality was demonstrated after the incorporation of PEG-mono-succimidyl-succinate (MSPEG), or PEG-di-succimidyl-succinate "end" capped with albumin (DSPEG) with or without gene transfer of Bcl-2. Islets treated with MSPEG presented a significant reduction in lactate dehydrogenase release compared with controls (41.2 +/- 3 vs 72.1 +/- 7, respectively, P < .05). Further protection was accomplished by DSPEG or AdBcl-2. The maximal cytoprotection was achieved by DSPEG +AdBcl-2 (15.5 +/- 4.9%, P < .001). Nonfasting glucose >200 mg/dL was found in 100% of the animals given control islets (n = 6) within 48 hours post-transplant. In contrast, euglycemia was achieved in 100 % of the animals given islets modified with DSPEG + AdBcl-2 during the observation time.Conclusions. Surface-engineering with functionalized PEG derivatives in combination with genetic modification with Bcl-2 significantly reduced islet loss after PIT. Application of this novel technology may improve results in xenoislet transplantation.