缺氧抑制胰岛β细胞中5-羟色胺生成的机制研究

Mechanism of hypoxia inhibition of serotonin production in pancreatic isletβ-cells in vitro

目的探讨缺氧条件下胰岛中5-羟色胺的表达水平及其调节机制。方法将体外培养的人胰岛分为对照组和氯化钴(CoCl_(2))处理组,每组设置3个平行组,通过免疫荧光染色检测人胰岛中的5-羟色胺表达水平。将β细胞系NIT-1细胞分为7组:对照组、CoCl_(2)处理组、缺氧(1%O_(2))组、缺氧诱导因子1α(Hif1α)激活剂二甲基乙二酰氨基乙酸(DMOG)组、小干扰RNA对照(sictrl)组、sictrl+CoCl_(2)组、siHif1α+CoCl_(2)组,每组设置3个平行组。通过实时荧光定量聚合酶链反应(qRT-PCR)和Western blotting检测5-羟色胺合成限速酶色氨酸羟化酶(Tph1)的表达水平。组间比较采用独立样本t检验。结果在人胰岛中,与对照组相比,CoCl_(2)处理组5-羟色胺的表达水平显著降低(P<0.001)。在NIT-1细胞中,与对照组相比,CoCl_(2)处理和缺氧处理(1%O_(2))均显著抑制Tph1表达(P<0.05),且Hif1α激活剂DMOG提高Hif1α表达(P<0.05),同时显著抑制Tph1表达(P<0.05);与siCtrl组相比,siCtrl+CoCl_(2)组Tph1表达显著下调(P<0.05),而siHif1α+CoCl_(2)组较siCtrl+CoCl_(2)组Tph1表达显著上调(P<0.05)。结论在缺氧条件下,Hif1α抑制Tph1的表达,从而降低胰岛中5-羟色胺的表达水平。

Objective To investigate the expression levels of serotonin in islets under hypoxic conditions and its regulatory mechanisms.Methods Human islets cultured in vitro were divided into control group and the hypoxia mimetic compound cobalt chloride(CoCl_(2))treatment group,and 3 parallel groups were set in each group.The expression levels of serotonin in human islets were detected through immunofluorescence staining.NIT-1 cells were divided into 7 groups:control group,CoCl_(2) group,hypoxia(1%O_(2))group,hypoxia inducible factor 1 alpha(Hif1α)activator dimethyloxalylglycine(DMOG)treatment group,small interfering RNA control(siCtrl)group,siCtrl+CoCl_(2) group and siHif1α+CoCl_(2) group,and 3 parallel groups were set in each group.Expression levels of the serotonin synthesis rate-limiting enzyme Tph1 were assessed by real-time quantitative polymerase chain reaction(qRT-PCR)and Western blotting.The independent sample t test was used to compare the results between two groups.Results The expression levels of serotonin significantly decreased in human islets treated with CoCl_(2) compared with control group(P<0.001).In NIT-1 cells,treatment with CoCl_(2) and hypoxia(1%O_(2))both significantly inhibited the expression of Tph1(P<0.05).The activation of Hif1αby DMOG increased the expression level of Hif1α(P<0.05),while simultaneously inhibiting the expression level of Tph1(P<0.05).Tph1 protein expression in siCtrl+CoCl_(2) group was significantly down-regulated than that in siCtrl group(P<0.05),but that in siHif1α+CoCl_(2) group was significantly up-regulated than that in siCtrl+CoCl_(2) group(P<0.05).Conclusion Under hypoxic conditions,Hif1αinhibits the expression of Tph1,thereby reducing the expression level of serotonin in the islets.