【作者】 王军；

【导师】 刘师伟；

【作者基本信息】 山西医科大学 ， 内科学， 2016， 硕士

【摘要】 目的:1、研究vaspin对棕榈酸诱导的INS-1胰岛β细胞的胰岛素抵抗和胰岛炎症的分子机制;2、研究vaspin是否可通过PI3K/Akt胰岛素信号通路和NF-κB炎症信号通路影响INS-1细胞的功能。方法:为了探讨vaspin对PI3K/Akt和NF-κB信号通路的影响以及确定合适的vaspin浓度,以INS-1细胞为研究对象分为5组:(1)对照组:完全培养基培养,不加干预因素;(2)PA(棕榈酸)组:0.5 mmol/l棕榈酸培养;(3)PA+vaspin(80 ng/ml)组:0.5 mmol/l棕榈酸+80 ng/ml的vaspin培养;(4)PA+vaspin(160 ng/ml)组:0.5 mmol/l棕榈酸+160 ng/ml的vaspin培养;(5)PA+vaspin(320 ng/ml)组:0.5 mmol/l棕榈酸+320 ng/ml的vaspin培养。Real-Time PCR检测上述5组细胞IRS-1、IRS-2、Akt、NF-κB的m RNA表达水平;Western blot检测IRS-2、p IRS-2、Akt、p Akt、NF-κB P65蛋白的表达水平。确定vaspin的合适浓度为320 ng/ml后,为了观察vaspin是否可通过激活PI3K/Akt信号通路介导INS-1细胞的分泌功能,实验分为3组:(1)对照组:0.5 mmol/l棕榈酸培养;(2)vaspin组:0.5 mmol/l棕榈酸+320 ng/ml的vaspin培养;(3)vaspin+ly294002(PI3K抑制剂)组:0.5 mmol/l棕榈酸+320 ng/ml的vaspin培养+25μmol/l的PI3K抑制剂ly294002培养。为了观察vaspin是否可通过抑制NF-κB信号通路介导INS-1细胞的分泌功能,以INS-1细胞为研究对象,并分为3组:(1)对照组:0.5 mmol/l棕榈酸培养;(2)vaspin组:0.5 mmol/l棕榈酸+320 ng/ml的vaspin培养;(3)TPCK(NF-кB抑制剂)组:作为阳性对照,0.5 mmol/l棕榈酸+20μmol/l的NF-кB抑制剂TPCK培养。Real-Time PCR检测上述分组细胞Akt、NF-κB的m RNA表达水平;Western blot检测Akt、p Akt、NF-κB P65蛋白的表达水平。ELISA检测葡萄糖刺激的胰岛β细胞分泌胰岛素的水平。结果:1.Vaspin对PI3K/Akt和NF-κB信号通路的影响与对照组相比,PA组IRS-2 m RNA、总的IRS-2蛋白、Akt的磷酸化水平(p Akt)和p Akt/Akt比例明显降低(P<0.05),NF-κB m RNA、NF-κB P65蛋白、IRS-2的磷酸化水平(p IRS-2)、p IRS-2/总IRS-2蛋白的比例明显增高(P<0.05)。和PA组相比,PA+vaspin(80 ng/ml)组、PA+vaspin(160 ng/ml)组、PA+vaspin(320 ng/ml)组使IRS-2 m RNA、总的IRS-2蛋白、p Akt和p Akt/Akt的比例明显上调和增高(P<0.05);NF-κB m RNA、NF-κB P65蛋白、p IRS-2和p IRS-2/总IRS-2蛋白的比例明显降低(P<0.05)。2.Vaspin对PI3K/Akt信号通路介导的INS-1胰岛β细胞功能的影响与对照组相比,vaspin组INS-1细胞p Akt/Akt的比例和葡萄糖刺激的INS-1细胞分泌的胰岛素水平明显增高(P<0.05)。而vaspin+ly294002(PI3K抑制剂)组和vaspin组相比,p Akt/Akt的比例和葡萄糖刺激的INS-1细胞分泌的胰岛素水平下降(P<0.05)。3.Vaspin对NF-κB信号通路介导的INS-1胰岛β细胞功能的影响与对照组相比,vaspin组和TPCK(NF-кB抑制剂)组INS-1细胞NF-κB m RNA和NF-κB P65蛋白的水平下调(P<0.05),葡萄糖刺激的INS-1细胞分泌的胰岛素水平明显增高(P<0.05)。与vaspin组相比,TPCK组NF-κB m RNA和NF-κB P65蛋白的水平增高(P<0.05),葡萄糖刺激的INS-1细胞分泌的胰岛素水平下降(P<0.05)。结论:棕榈酸可诱导胰岛β细胞产生胰岛素抵抗和炎症,vaspin激活PI3K/Akt信号通路和抑制NF-κB信号通路改善棕榈酸诱导的胰岛β细胞胰岛素抵抗和抑制胰岛β细胞炎症,并改善葡萄糖刺激的胰岛β细胞的分泌功能。本课题为国家自然科学基金“Vaspin在胰岛β细胞炎症、胰岛素抵抗及氧化应激中的作用及机制研究(编号:81471025)”。更多还原

【Abstract】 Objective:1. To investigate the mechanisms of vaspin induced insulin resistance and inflammation in INS-1 islet beta cells.2. To determine whether vaspin modulates the function of INS-1 cells through PI3K/Akt and NF-kappa B signaling pathways. Methods:In order to study the effects of vaspin on PI3K/Akt and NF-kappa B signaling pathways and determine the appropriate concentration of vaspin, INS-1 cells were cultured and divided into five groups:(1)the control group: treated with culture medium only;(2) the PA group: treated with 0.5 mmol/l palmitic acid;(3) PA + vaspin(80 ng/ml) group: treated with 0.5 mmol/l palmitic acid and 80 ng/ml vaspin;(4) PA + vaspin(160 ng/ml) group: treated with 0.5 mmol/l palmitic acid and 160 ng/ml vaspin;(5) PA + vaspin(320 ng/ml) group: treated with 0.5 mmol/l palmitic acid and 320 ng/ml vaspin. The m RNA levels of IRS-1, IRS-2, Akt, NF-κB were determined by Real-Time PCR. The protein levels of IRS-2, p IRS-2, Akt, p Akt, NF-κB P65 were determined by western blot.In order to determine whether vaspin improve insulin secretion of INS-1 cells through PI3K/Akt and NF-κB signaling pathways, INS-1 cells were divided into three groups respectively:(1) the control group: treated with 0.5 mmol/l palmitic acid;(2) vaspin groups: treated with 0.5 mmol/l palmitic acid and 320 ng/ml vaspin;(3) vaspin + ly294002(PI3K inhibitor) or TPCK(NF-κB inhibitor): treated with 0.5 mmol/l palmitic acid, 320 ng/ml vaspin and 25 μmol/l PI3 K inhibitors ly294002 or 20 μmol/l NF-κB inhibitors TPCK. The m RNA levels of Akt and NF-κB were determined by Real-Time PCR. The protein levels of Akt, p Akt, NF-κB P65 were determined by western blot. The glucose stimulated insulin secretion(GSIS) were determined by ELISA. Results:1. The effects of vaspin on PI3K/Akt and NF-κB signaling pathwaysCompared with control group, PA significantly decreased the level of IRS-2 m RNA, total IRS-2 protein and Akt phosphorylation, while increased the NF-κB m RNA and NF-κB P65 protein levels and the IRS-2 phosphorylation levels(P<0.05). Pretreatment of cells with different concentration of vaspin reversed these PA treatment related effects(P<0.05).2. The effects of vaspin on PI3K/Akt signaling pathway induced by PAAkt phosphorylation and GSIS levels were significantly increased after treatment with vaspin compared to the control group(P<0.05). Pretreatment with PI3 K inhibitor ly294002 blocked this effect of vaspin(P<0.05).3. The effects of vaspin on NF-κB signaling pathway induced by PACompared with control group, vaspin and NF-кB inhibitor, TPCK significantly decreased the m RNA and protein levels of NF-κB and GSIS(P< 0.05). ConclusionVaspin protects the islet beta cells from palmitic acid induced insulin resistance and inflammation through PI3K/Akt and NF-κB signaling pathways.更多还原