肿瘤坏死因子α（TNF—α）对SD大鼠胰岛β细胞凋亡的分子机制

The Molecular Mechanism of Apoptosis of Rat Pancreatic Islet β- Cells Induced by Various Concentration Tumor Necrosis Factor

目的 探讨TNF—α对体外SD大鼠胰岛β细胞调亡的分子机制。方法 成年雄性SD大鼠胰岛细胞经胶原酶P消化，Ficoll400纯化后，在37℃，5％CO2，RPMI1640培养剂培养7d，铺成单层，用不同浓度TNF-α（0、10、30、50ng／mL）孵育24h。应用放免法测定不同浓度上清夜及葡萄糖刺激时上清液中胰岛素水平；应用RT—PCR检测胰岛细胞Bcl-2和Bax mRNA表达水平。结果 随TNF-α浓度的增加：（1）胰岛β细胞葡萄糖刺激的胰岛素分泌反应逐渐降低；（2）Bax mRNA表达水平表达呈稳步上升趋势，明显高于对照组（P〈0．05）；而Bcl-2mRNA表达水平在10ng／mL时轻度上升．随着剂量的加大（30-50ng/mL）表达开始明显下降，明显低于对照组（P〈0．05）。结论 （1）TNF-α可诱导大鼠胰岛细胞功能缺陷。（2）大鼠胰岛细胞的Bcl-2和Bax mRNA表达水平变化在TNF—α所致的胰岛细胞凋亡中起重要作用。

Objective To investigate the role of various tumor necrosis factor- α in apoptosis of rat pancreatic islet β - cells and its molecular mechanism. Methods Islets were isolated from male adult sprague - dawley rats （200 - 250 g）and digested for 10 min at 38℃ by collagenase ,and then were separated on a Ficon step density gradient and were cultured for7 days in an atmosphere of 95% air, 5% CO2 at 37℃. When the islet cells flatted to monolayer, they were divided into different concentration TNF- α （0, 10,30,50 ng/mL） group and cultured for 24 hours respectively, the levels of insulin in supematant of cultured islet cells of various concentration group and response of glucose stimulating insulin secretion （GSIS） were studied by RIA,and the mRNA levels of genes of Bcl- 2 and Bax in these cells culture by above condition were detected by semiquantitive RT- PCR. Results With the increase of concentrations of tumor necrosis factor - α： （ 1 ） the GSIS in rat islet cells was markedly decreased than those of normal control group; （2） the levels of Bax mRNA were significantly higher than those of control group in dose dependently （ P 〈 0.05）, while the levels of Bcl - 2 mRNA slightly rose at concentration of 10 ng/mL and then significantly decreased at 30 - 50 ng/mL as compared with those of control group （ P 〈 0.05）. Conclusions （ 1 ） In cultured circumstances in vitro,TNF- α is toxic to rat islet cells,as manifested by the dysfunction of GSIS. （2） The expression change of Bax and Bcl - 2 seems to he an important predictor of islet cells apoptosis.