用改良的 EGM2 培养基培养可增强猪胰岛样细胞簇在胰岛异种移植中的抗凋亡能力。

Culturing with modified EGM2 medium enhances porcine neonatal islet-like cell clusters resistance to apoptosis in islet xenotransplantation.

机构信息

Cell Transplantation and Gene Therapy Institute, The Third Xiangya Hospital, Central South University, Changsha, Hunan, China.

Engineering and Technology Research Center for Xenotransplantation of Human Province, Changsha, Hunan, China.

出版信息

Xenotransplantation. 2018 Jan;25(1). doi: 10.1111/xen.12358. Epub 2017 Nov 12.

DOI:10.1111/xen.12358

PMID:29131417

Abstract

BACKGROUND

Neonatal pig islet-like cell clusters (NICC) are an attractive source of insulin-producing tissue for potential transplantation treatment of type 1 diabetic patients. However, a considerable loss of NICC after their transplantation due to apoptosis resulted from islet isolation and instant blood-mediated inflammatory reaction remains to be overcome.

METHODS

EGM2 medium depleted with hydrocortisone and supplemented with 50 mmol/L isobutylmethylxanthine, 10 mmol/L nicotinamide, and 10 mmol/L glucose was used to culture NICC at day 1, the day after isolation and changed every other day. NICC cultured with EGM2 or control Ham's F-10 medium were collected at day 7 of culture for the following assays. The viability of NICC was evaluated by AO/EB staining and FACS. Static assay and oxygen consumption rate analysis were performed to assess the function of NICC. Insulin and glucagon gene expression were measured by real-time PCR. Tubing loops model and TUNEL assay were performed to confirm the apoptosis-resistant ability of NICC cultured with modified EGM2 medium. Serum starvation and hypoxia treatment were used to test the tolerant capability of NICC in the microenvironment of hypoxia/nutrient deficiency in vitro. The molecules involved in apoptosis pathways in NICC were analyzed by Western blotting.

RESULTS

Compared with Ham's F-10 medium, culturing NICC with EGM2 medium led to increased number and viability of NICC with higher stimulation index, upregulated gene expression of both insulin and glucagon, and enhanced mitochondria function. Furthermore, fewer modified EGM2 medium cultured NICC were found under apoptosis when evaluated in an in vitro tubing loop model of IBMIR. Moreover, EGM2 medium cultured NICC demonstrated much less apoptotic cells under either serum starvation or hypoxia condition than their Ham's F-10 medium cultured counterparts. The enhanced capability of EGM2 cultured NICC to resist apoptosis was associated with their elevated protein levels of anti-apoptotic Bcl-2 family member Mcl-1.

CONCLUSION

Culturing NICC with EGM2 provides a simple and effective approach not only to increase NICC yield, viability, and maturation but also to enhance their resistance to apoptosis to preserve the initial graft mass for successful islet xenotransplantation.

摘要

背景

新生猪胰岛样细胞簇（NICC）是胰岛素产生组织的有吸引力的来源，可用于潜在的 1 型糖尿病患者的移植治疗。然而，由于胰岛分离和即时的血液介导的炎症反应导致的细胞凋亡，NICC 在移植后会有相当大的损失，这一问题亟待解决。

方法

使用 EGM2 培养基培养 NICC，该培养基中含有去氢皮质酮和 50mmol/L 的异丁基甲基黄嘌呤、10mmol/L 的烟酰胺和 10mmol/L 的葡萄糖，在第 1 天、分离后的第 2 天和每隔一天培养 NICC。在第 7 天培养时，收集用 EGM2 或对照 Ham's F-10 培养基培养的 NICC，进行以下检测。通过吖啶橙/溴乙锭（AO/EB）染色和流式细胞术评估 NICC 的活力。进行静态测定和耗氧量分析以评估 NICC 的功能。通过实时 PCR 测量胰岛素和胰高血糖素基因的表达。使用管状环模型和 TUNEL 测定法来确认在改良的 EGM2 培养基中培养的 NICC 的抗凋亡能力。通过血清饥饿和缺氧处理来测试 NICC 在体外缺氧/营养缺乏的微环境中的耐受能力。通过 Western blot 分析 NICC 中凋亡途径相关的分子。

结果

与 Ham's F-10 培养基相比，用 EGM2 培养基培养 NICC 可增加 NICC 的数量和活力，提高刺激指数，上调胰岛素和胰高血糖素的基因表达，并增强线粒体功能。此外，在 IBMIR 的体外管状环模型中，改良的 EGM2 培养基培养的 NICC 凋亡细胞数量更少。此外，与 Ham's F-10 培养基培养的 NICC 相比，在血清饥饿或缺氧条件下，用 EGM2 培养基培养的 NICC 的凋亡细胞更少。用 EGM2 培养基培养的 NICC 增强的抗凋亡能力与其抗凋亡 Bcl-2 家族成员 Mcl-1 的蛋白水平升高有关。