胎球蛋白A的纯化及其对3T3-L1脂肪细胞胰岛素通路的抑制作用  

Purification of AHSG and its inhibition effect on insulin pathway of 3T3-L1 Adipocyte

作　　者：陈婷[1] 靳彦文[1] 瞿秀华[1] 崔艳[1] 李大培[1] 何章荣[1] 朱晓辉[1] 曹诚[1] 

机构地区：[1]军事医学科学院生物工程研究所,北京100850

出　　处：《解放军医学杂志》2010年第7期819-822,共4页Medical Journal of Chinese People's Liberation Army

基　　金：国家高技术研究发展计划(863计划)项目(2005AA218080)

摘　　要：目的对胎球蛋白A(AHSG)蛋白进行纯化,并探讨其对脂肪细胞胰岛素通路的调节作用。方法通过硫酸铵沉淀、PEG沉淀、DEAE离子交换和Protamine亲和层析方法纯化血浆中的AHSG蛋白。培养3T3-L1前脂肪细胞并分化为成熟脂肪细胞,置于低糖DMEM培养基培养,然后加入终浓度为100μg/ml和600μg/ml的AHSG及终浓度为600μg/ml的人白蛋白(HAS)分别进行培养,最后用不同浓度(0、10、20nmol/L)的胰岛素刺激细胞。采用Western blotting检测AHSG和HSA对3T3-L1脂肪细胞胰岛素下游分子IRS-1和Akt的磷酸化水平的影响。结果从血浆中纯化的AHSG蛋白纯度达90%以上。纯化的AHSG在100μg/ml浓度下可以减弱胰岛素对IRS-1蛋白磷酸化的影响,在600μg/ml浓度下可以完全抑制IRS-1蛋白磷酸化;纯化的AHSG在100μg/ml浓度下可以减弱胰岛素对Akt蛋白磷酸化的影响,在600μg/ml的浓度下可以明显抑制Akt蛋白的磷酸化,而600μg/ml的HAS则不能抑制IRS-1和Akt蛋白的磷酸化。结论从血清中纯化出的AHSG在生理浓度下可以抑制胰岛素对3T3-L1细胞中IRS-1和Akt蛋白的磷酸化作用,在脂肪细胞胰岛素通路中扮演着重要角色。Objective To purify α 2-Heremans-Schmid glycoprotein （AHSG） from plasma and optimize the condition for purification,and to explore the effects of AHSG on the regulation of insulin pathway of 3T3-L1 adipocytes by detecting IRS-1 and Akt protein phosphorylation.Methods AHSG was isolated from plasma,and purified by precipitation with ammonium sulfate and also with PEG,DEAE Sepharose fast flow anion exchange chromatography and protamine-agarose Affinity chromatography.The effects of AHSG on phosphorylation of IRS-1 and Akt protein in 3T3-L1 adipocytes were tested with Western blotting.3T3-L1 adipocytes differentiated from the preadipocytes were cultured with low-sugar DMEM medium without serum,and then pretreated with AHSG at a low concentration of 100μg/ml,then a physiological concentration of 600μg/ml,and with human serum albumin （HSA） at a concentration of 600μg/ml.The adipocytes were then stimulated by different concentrations of insulin,and phosphorylation of IRS-1 and Akt protein in treated adipocytes were assayed with Western blotting.Results The purity of AHSG purified from plasma reached 90% and above.The phosphorylation of IRS-1 and Akt protein in 3T3-L1 adipocytes was stimulated by insulin.Insulin-stimulated phosphorylation of IRS-1 protein in 3T3-L1 adipocytes was attenuated by purified AHSG in concentration of 100μg/ml,and completely inhibited by purified AHSG in concentration of 600μg/ml;the insulin-stimulated phosphorylation of Akt protein in 3T3-L1 adipocytes was attenuated by purified AHSG in concentration of 100μg/ml,and obviously inhibited by purified AHSG in concentration of 600μg/ml;while the insulin-stimulated phosphorylation of Akt and IRS-1 protein in 3T3-L1 adipocytes could not be inhibited by HSA in concentration of 600μg/ml.Conclusion Insulin-stimulated phosphorylation of IRS-1 and Akt protein in 3T3-L1 adipocytes can be inhibited by AHSG in a physiological concentration,implying that AHSG plays an important role in insulin pathway of 3T3-L1 adipocytes.

关 键 词：胎球蛋白A 胰岛素 受体 胰岛素 蛋白激酶类 

分 类 号：R153.2[医药卫生—营养与食品卫生学] Q786[医药卫生—公共卫生与预防医学]