Eckhoff D E, Sollinger H W, Hullett D A
Department of Surgery, University of Wisconsin School of Medicine, Madison 53792.
Transplantation. 1991 Jun;51(6):1161-5. doi: 10.1097/00007890-199106000-00004.
A limited collagenase digestion was used to prepare human fetal pancreatic (HFP) proislets that maintained in vitro and in vivo functional viability. Digestion times, collagenase concentrations, and culture conditions were optimized by assessing insulin release in response to low glucose (3.3 mM), high glucose (16.7 mM), and high glucose plus theophylline (10 mM). A stimulation index (ng insulin release in low glucose/ng insulin in high glucose plus theophylline) and insulin production (ng insulin/mg tissue) were used to determine functional viability. A media change 24 hr after digestion resulted in a marked increase in mean stimulation index (6.18 +/- 1.2 vs. 3.12 +/- 0.9). IGF-1 was found to enrich beta-cell viability as demonstrated by insulin-specific immunoperoxidase staining and insulin release in response to HGT (36.3 +/- 7.7 ng/mg tissue vs. 18.1 +/- 5.2 ng/mg tissue). Immunohistologic staining of proislets suggested selective enrichment of beta cells. Diabetic BALB/c/nu/nu transplanted with proislets cultured in the presence of IGF-1 (100 ng/ml) returned to normoglycemia in 6.7 +/- 3.1 weeks (range 4-11) versus 12.0 +/- 4.0 (range 10-15) in controls. Oral glucose tolerance test demonstrated in vivo serum glucose control equivalent to nondiabetic control mice. These findings suggest that HFP proislet preparation and culture with endocrine growth factors, specifically IGF-1, may provide a source of tissue with enriched beta-cell activity that may have decreased immunogenic load and is more suitable for clinical transplantation.
采用有限胶原酶消化法制备人胎儿胰腺（HFP）胰岛前体细胞，这些细胞在体外和体内均保持功能活性。通过评估低葡萄糖（3.3 mM）、高葡萄糖（16.7 mM）以及高葡萄糖加茶碱（10 mM）刺激后的胰岛素释放情况，对消化时间、胶原酶浓度和培养条件进行了优化。使用刺激指数（低葡萄糖刺激下的胰岛素释放量（ng）/高葡萄糖加茶碱刺激下的胰岛素释放量（ng））和胰岛素产量（胰岛素含量（ng）/组织质量（mg））来确定功能活性。消化后24小时更换培养基，平均刺激指数显著增加（6.18±1.2对3.12±0.9）。胰岛素特异性免疫过氧化物酶染色和对高糖刺激（HGT）的胰岛素释放结果表明，胰岛素样生长因子-1（IGF-1）可增强β细胞活性（组织胰岛素含量为36.3±7.7 ng/mg对18.1±5.2 ng/mg）。胰岛前体细胞的免疫组织化学染色显示β细胞选择性富集。移植了在IGF-1（100 ng/ml）存在下培养的胰岛前体细胞的糖尿病BALB/c/nu/nu小鼠在6.7±3.1周（范围4 - 11周）恢复正常血糖，而对照组为12.0±4.0周（范围10 - 15周）。口服葡萄糖耐量试验表明其体内血清葡萄糖控制情况与非糖尿病对照小鼠相当。这些发现提示，HFP胰岛前体细胞的制备以及与内分泌生长因子（特别是IGF-1）共培养，可能提供一种具有增强β细胞活性的组织来源，其免疫原性负荷可能降低，更适合临床移植。
