中文摘要：

【目的】研究褪黑素对大鼠胰岛瘤细胞INS-1中胰岛素和Gαi/o蛋白基因表达的影响,探究褪黑素在m RNA水平对Gαi/o和Insulin1调节作用中可能存在的分子机制。褪黑素（melatonin,MT）是松果腺分泌的一种吲哚类神经内分泌激素,在机体内可调节胰岛素的分泌而使其呈现昼夜节律性分泌,影响葡萄糖昼夜代谢水平的变化进而维持机体血糖的相对恒定,故对于褪黑素影响胰岛素表达的研究可能会对褪黑素在胰岛素昼夜节律性分泌中的调控作用提供依据。【方法】将冻存的INS-1细胞复苏培养至第三代,INS-1细胞传代后,用RPMI1640培养基培养INS-1细胞约24—48 h,当细胞密度达60%—70%时,使用无血清无葡萄糖的RPMI1640洗2次,然后在INS-1细胞培养外液中分别加入不同浓度（0、10、20、30、50 mmol·L-1）的葡萄糖及100 nmol·L-1褪黑素和不同浓度葡萄糖孵育INS-1细胞12 h,观察和统计INS-1细胞的形态学变化。利用Easy Pure？RNA Kit提取INS-1细胞的总RNA,核酸蛋白测定仪测定细胞总RNA的浓度和纯度,其次利用甲醛变性琼脂糖凝胶电泳检测细胞的总RNA质量,Trans Script One-Step g DNA Removal and c DNA Synthesis Super Mix反转录合成c DNA。利用Primer Premier5.0引物设计软件,参考Gen Bank上已登录的基因序列进行Insulin1、Gαi1、Gαi2、Gαo、PKA和PKCα引物的设计。应用q RT-PCR法进行检测处理后的INS-1细胞Insulin1、Gαi1、Gαi2、Gαo、PKA和PKCαm RNA水平的变化。【结果】用含不同浓度葡萄糖培养液孵育INS-1细胞后,观察发现随着培养基中葡萄糖浓度含量的增加INS-1细胞突触的增长呈现先增加后减少的趋势,其中20 mmol·L-1葡萄糖处理组INS-1细胞突触的增长明显,而20 mmol·L-1葡萄糖和100 nmol·L-1褪黑素处理组INS-1细胞表面积增大,但突触增长却不明显;培养基中含不同浓度葡萄糖的处理组中,Insulin1 m RNA水平呈现先升后降的趋势,Gαi1 m RNA水平则呈

英文摘要：

【Objective】To demonstrate the role of melatonin in the circadian secretion of insulin, the effect of melatonin on insulin and Gαi/o expression in rat insulinoma cell line（INS-1） and molecular mechanism were studied. As melatonin can regulate insulin secretion in a circadian manner, maintaining a homeostasis of blood glucose by regulating diurnal glucose metabolism, thus study on the effect of melatonin on expression of insulin may provide a basis for regulation of melatonin in circadian secretion of insulin.【Method】The cryopreserved INS-1 cells were passaged for at least three generations. After the passage of cells, INS-1 cells were cultured in RPMI1640 medium for about 24-48 h. When the cell confluency reached 60%-70%, washed with RPMI1640 without serum and glucose for two times. INS-1 cells were cultured in media supplemented with different concentrations of glucose（0, 10, 20, 30, and 50 mmol·L-1） and 100 nmol·L-1 melatonin for 12 h treatment, then observation of morphological changes of INS-1 cells and statistical analysis were performed. The total RNA extracted from INS-1 cells by Easypure RNA kit, measuring the cells total RNA concentration and purity, then the quality of total RNA was measured by formaldehyde denaturing agarose gel electrophoresis. Tran Script One-step Removal and Synthesis Supermix were used to synthesize c DNA by reverse transcription. Primer Premier 5.0 primer design software, reference gene sequence of Gen Bank were used to design primers. q RT-PCR was used to detect the changes of Insulin1, Gαi1, Gαi2, Gαo, PKA and PKCα in m RNA level.【Result】After incubation of INS-1 cells with different concentrations of glucose, it was observed that the growth of cell synapse first increased and then decreased along with the increase of the concentration of glucose in the culture medium. In the 20 mmol·L-1 glucose treatment group, the synaptic growth of INS-1 cells was increased significantly; in the 20 mmol·L-1glucose and melatonin co-treatment group, the area of INS-1