Development of a novel β-cell specific promoter system for the identification of insulin-producing cells in in vitro cell cultures
Development of a novel β-cell specific promoter system for the identification of insulin-producing cells in in vitro cell cultures
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DOI：
10.1016/j.yexcr.2006.07.015
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发表时间：
2006-10-15
影响因子：
3.7
通讯作者：
Tanaka, Noriaki
中科院分区：
文献类型：
--
作者：
Fukazawa, Takuya;Matsuoka, Junji;Tanaka, Noriaki
最近，有报道称，对1型糖尿病患者进行胰岛移植可以实现一年或更长时间的胰岛素独立[Shapiro等人，使用非糖皮质激素免疫抑制方案N Engl J Med对7名1型糖尿病患者进行胰岛移植。343(2000)230-238]。然而，供体胰岛组织的数量是有限的，因此，人们正在探索多种方法来在体外产生胰岛素产生细胞。利用小鼠和人类干细胞和祖细胞已经获得了一些有希望的结果[Soria等人，从干细胞到β细胞：糖尿病细胞治疗的新策略。4(2001)407-415；Lechner等人，来自成人组织的干细胞/祖细胞：治疗糖尿病的潜力，Am J Physiol Endocrinol Metab。第284(2003)259-266；Bonner-Weir等人，从扩张的导管组织中体外培养人的胰岛，《中华儿科杂志》，S A，97(2000)7999-8004；阿萨迪等，《人类胚胎干细胞产生胰岛素》，50(2001)糖尿病，1691-1697]。然而，获得大量分化细胞的群体的效率一直很低。为了提高从未分化细胞中生产和筛选胰岛素分泌细胞的效率，我们设计了一个新的β细胞特异性和葡萄糖反应启动子系统，命名为pGL3.hINS-363x。这个人工启动子系统不仅在产生胰岛素的MIN6 M9细胞中显示出显著的荧光素酶活性，而且在分离的人胰岛中也显示出显著的荧光素酶活性。PGL3.hINS-363x在低糖(2 mM葡萄糖)条件下无活性，但在高糖(16 mM葡萄糖)条件下表现出显著的活性和β细胞特异性。此外，pGL3.hINS-363 3x对分化的AR42J细胞具有显著的启动子活性，经激活素A和β细胞蛋白处理后，AR42J细胞可以产生胰岛素。在这里，我们描述了一种新的β细胞特异性和葡萄糖响应型人工启动子系统，旨在分析和分类从干细胞或其他祖细胞分化而来的类似β胰岛素的细胞。(C)2006 Elsevier Inc.保留所有权利。
Recently, it has been reported that islet transplantation into patients with Type 1 diabetes may achieve insulin independence for a year or longer [Shapiro et al., Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen, N Engl J Med. 343 (2000) 230-238]. However, the amount of donor islet tissue is limited, therefore, multiple approaches are being explored to generate insulin-producing cells in vitro. Some promising results have been obtained using mouse and human stem cells and progenitor cells [Soria et al., From stem cells to beta cells: new strategies in cell therapy of diabetes mellitus, Diabetologia. 4 (2001) 407-415; Lechner et al., Stem/progenitor cells derived from adult tissues: potential for the treatment of diabetes mellitus, Am J Physiol Endocrinol Metab. 284 (2003) 259-266; Bonner-Weir et al., In vitro cultivation of human islets from expanded ductal tissue, Proc Natl Acad Sci U S A, 97 (2000) 7999-8004; Assady et al., Insulin production by human embryonic stem cells, 50 (2001) Diabetes 1691-1697]. However, the efficiency of obtaining populations with high numbers of differentiated cells has been poor. in order to improve the efficiency of producing and selecting insulin-producing cells from undifferentiated cells, we have designed a novel beta-cell specific and glucose responsive promoter system designated pGL3.hINS-363 3x. This artificial promoter system exhibits significant luciferase activity not only in insulin-producing MIN6 m9 cells but also in isolated human islets. The pGL3.hINS-363 3x construct shows no activity in non-insulin-producing cells in low glucose conditions (2 mM glucose) but demonstrates significant activity and beta-cell specificity in high glucose conditions (16 mM glucose). Furthermore, pGL3.hINS-363 3x shows significant promoter activity in differentiated AR42J cells that can produce insulin after activin A and betacellulin treatment. Here, we describe a novel beta-cell specific and glucose responsive artificial promoter system designed for analyzing and sorting beta-like insulin-producing cells that have differentiated from stem cells or other progenitor cells. (c) 2006 Elsevier Inc. All rights reserved.