Thivolet C H, Demidem A, Haftek M, Durand A, Bertrand J
Institut National de la Santé et de la Recherche Medicale (INSERM), U34, Hôpital Debrousse, Lyon, France.
Diabetes. 1988 Sep;37(9):1279-86. doi: 10.2337/diab.37.9.1279.
Dissociated human insulinoma cells were plated onto plastic multiwell dishes. Cells were maintained for 1 mo on plastic with three passages. Cultures consisted of small colonies with some areas of stratification and few intercellular spaces. Ultrastructural studies indicated that cultured cells had epithelial features with desmosomes at cell-to-cell contacts and intermediate filaments in addition to secretory granules in the cytoplasm. Insulin and C-peptide were released in equimolar amounts in culture media. When challenged for 30 min with 16.7 mM glucose, 1 mM 3-isobutyl-1-methylxanthine, 4 mM tolbutamide, or 10(-6) M glucagon, insulinoma cells responded by a 1.5-, 1.5-, 2-, or 3-fold increase, respectively, in insulin release above baseline levels. A 15-min challenge with 10(-5) M isoproterenol increased insulin secretion by 1.85-fold. By indirect immunofluorescence, an anti-insulin antibody reacted positively with cell cytoplasm, whereas anti-somatostatin and anti-glucagon antibodies did not. Insulinoma cell surface expressed class I MHC molecules but not class II molecules. Immediately after isolation, crude insulinoma cells were contaminated by 2% of DR+ cells from nonislet components that disappeared after several weeks in culture. The ability of insulinoma cells to stimulate allogenic T-lymphocyte proliferation was assessed by [3H]thymidine incorporation in mixed culture combinations. Crude insulinoma cells elicited a strong lymphoproliferative response with a stimulation index ranging between 3.5 and 7, whereas no stimulation was found after 1 mo in culture. It is postulated that absence of class II-positive cells in the stimulatory cell preparation conditioned this immune tolerance across the major histocompatibility barrier.(ABSTRACT TRUNCATED AT 250 WORDS)
将解离的人胰岛素瘤细胞接种到塑料多孔培养皿中。细胞在塑料培养皿上培养1个月，传代3次。培养物由一些有分层区域和少量细胞间隙的小菌落组成。超微结构研究表明，培养的细胞具有上皮细胞特征，细胞间连接处有桥粒，细胞质中有中间丝，此外还有分泌颗粒。胰岛素和C肽以等摩尔量释放到培养基中。当用16.7 mM葡萄糖、1 mM 3 -异丁基-1 -甲基黄嘌呤、4 mM甲苯磺丁脲或10(-6) M胰高血糖素刺激30分钟时，胰岛素瘤细胞的胰岛素释放量分别比基线水平增加1.5倍、1.5倍、2倍或3倍。用10(-5) M异丙肾上腺素刺激15分钟可使胰岛素分泌增加1.85倍。通过间接免疫荧光法，抗胰岛素抗体与细胞质呈阳性反应，而抗生长抑素和抗胰高血糖素抗体则无此反应。胰岛素瘤细胞表面表达I类主要组织相容性复合体分子，但不表达II类分子。刚分离后，粗制胰岛素瘤细胞被来自非胰岛成分的2%的DR +细胞污染，这些细胞在培养几周后消失。通过在混合培养组合中掺入[3H]胸腺嘧啶核苷来评估胰岛素瘤细胞刺激同种异体T淋巴细胞增殖的能力。粗制胰岛素瘤细胞引发强烈的淋巴细胞增殖反应，刺激指数在3.5至7之间，而培养1个月后未发现刺激作用。据推测，刺激细胞制剂中缺乏II类阳性细胞导致了这种跨越主要组织相容性屏障的免疫耐受。（摘要截断于250字）
