【作者】 韩冠军；

【导师】 朱大龙；

【作者基本信息】 南京医科大学 ， 内科学， 2009， 硕士

【摘要】 目的:急性时相血清淀粉样蛋白A(Acute phase serum amyloid protein A, A-SAA)是近年来研究比较多的脂源性细胞因子,其在肥胖(obesity)和2型糖尿病(Type 2 Diabetes Mellitus)等慢性炎症患者血清中表达轻度升高。我们近年研究发现人重组A-SAA能够显著促进ECV304内皮细胞和人脐静脉内皮细胞(HUVECs)IL-6、IL-8 mRNA表达,且呈时间和剂量依赖性,但A-SAA对脂肪细胞炎症因子表达的影响目前尚不明确。本研究旨在探讨急性时相血清淀粉样蛋白A(A-SAA)对3T3-L1脂肪细胞因子白细胞介素-6(Interleukin-6,IL-6)、肿瘤坏死因子-a (Tumor necrosis factor,TNF-α)和脂联素(adiponectin)基因、蛋白及相关激酶表达的影响。方法:体外培养、诱导分化3T3-L1前脂肪细胞(3T3-L1 preadipocytes),分别用不同浓度的人重组A-SAA(0μg/ml、1μg/ml、10μg/ml、20μg/ml),在不同时间段(0、8h、24h、48h),干预分化成熟的3T3-L1脂肪细胞,用半定量逆转录聚合酶链反应(Reverse transcription Polymerase Chain Reaction,RT-PCR)检测A-SAA对IL-6、TNF-α和adiponectin mRNA表达的影响;用酶联免疫吸附法(Enzyme-linked Immunosorbent Assay,ELISA)检测条件培养基中上述脂肪细胞因子的表达变化;用免疫印迹法(Western Blot)检测A-SAA对相关激酶C-Jun氨基末端激酶( c-jun N-terminal kinase, JNK)和细胞外信号调节激酶(Extracellular signal-regulated kinase ,ERK)蛋白表达的影响。结果:1、与对照组比较,10μg/ml A-SAA和20μg/ml A-SAA分别干预8h后IL-6 mRNA的表达均显著升高(P<0.05和P<0.01),且20μg/ml A-SAA干预8h组该基因的表达较1μg/ml A-SAA干预8h组也显著升高(P<0.05);与对照组比较,三种浓度的A-SAA分别干预24h后IL-6 mRNA的表达均显著升高(P<0.01),且20μg/ml A-SAA干预24h组该基因的表达较1μg/ml A-SAA干预24h组也显著升高(P<0.01);分别与对照组比较,1μg/ml A-SAA和10μg/ml A-SAA分别干预不同时间后各组IL-6 mRNA表达均显著升高(P<0.05和P<0.01);与对照组比较,20μg/ml A-SAA干预8h组和干预24h组IL-6 mRNA表达显著升高(P<0.01),且20μg/ml A-SAA干预8h组较干预48h组也显著升高(P<0.05);2、与对照组比较,10μg/ml A-SAA干预24h后TNF-a mRNA表达显著升高(P<0.05);3、10μg/ml A-SAA干预24h,使3T3-L1细胞adiponectin mRNA的表达显著下降(P<0.05);4、不同浓度A-SAA干预不同时间后培养基中的TNF-a、IL-6和adiponectin的表达均没有显著变化(P均﹥0.05);5、A-SAA可能激活JNK和ERK。结论:A-SAA剂量依赖性地促进IL-6 mRNA的表达,10μg/ml A-SAA干预24h使TNF-a和IL-6 mRNA表达显著升高,同时显著降低adiponectin mRNA的表达;A-SAA对条件培养基中脂肪细胞因子蛋白的表达没有影响;A-SAA没有通过促进TNF-a和IL-6表达和(或)降低脂联素表达间接引起胰岛素抵抗,A-SAA可能通过激活MAPK信号通路,参与脂肪细胞的胰岛素抵抗。更多还原

【Abstract】 Objectives:Acute phase serum amyloid protein A (A-SAA) is one of the frequently studied adipocytokines recently, which present mild increase in obesity and T2DM. Our previous studies showed that human recombinated A-SAA could significantly induce ECV304 endotheliocytes and human umbilical vein endothelial cells,(HUVECs)IL-6、IL-8 mRNA expression in a dose- and time-dependent manner.However,whether A-SAA will effect the expression of inflammatory cytokines in adipocytes is unclear. Our aims are to examine the roles of acute phase serum amyloid protein A in regulating adipocytokines and related kinases expression in 3T3-L1 adipocytes in this study.Methods:3T3-L1 preadipocytes cultured and differentiated in six-well plates containing high glucose DMEM plus 10% fetal calf serum according to Kolapo M Ajuwon were treated with 3 different concentrations of human recombinated A-SAA(1 ug/ml、10 ug/ml、20 ug/ml) for 3 time periods(8h、24h、48h)after preadipocytes fully differentiated. IL-6、TNF-a and adiponectin mRNA expressions were detected by Reverse transcription Polymerase Chain Reaction(RT-PCR), the concentrations of TNF-a、IL-6 and adiponectin in conditioned media were tested by Enzyme-linked Immunosorbent Assay( ELISA), and the phosphorylated protein expressions of JNK and ERK were detected by Western Blot.Results:Compared with control group, both 10μg/ml A-SAA and 20μg/ml A-SAA significantly induced the IL-6 mRNA expression respectively after incubated for 8h(P<0.05 and P<0.01)and compared with 1μg/ml A-SAA group, 20μg/ml A-SAA also significantly induced the IL-6 mRNA expression(P<0.05);compared with control group, 3 different concentrations of A-SAA (1 ug/ml、10 ug/ml、20 ug/ml) significantly induced the IL-6 mRNA expression respectively after incubated for 24h(P<0.01)and compared with 1μg/ml A-SAA group, 20μg/ml A-SAA also significantly induced the IL-6 mRNA expression(P<0.01);compared with control group,both 1μg/ml A-SAA and 10μg/ml A-SAA also significantly induced the IL-6 mRNA expression respectively after incubated for different times (8h、24h、48h)(P<0.05 and P<0.01);compared with control group,20μg/ml A-SAA also significantly induced the IL-6 mRNA expression after incubated for 8h and 24h ( P <0.01);compared with control group,the mRNA expression of adiponectin was significantly decreased after stimulated with 10μg/ml A-SAA for 24h(P<0.05), the mRNA expression of TNF-a was significantly increased after treated with 10μg/ml A-SAA for 24h(P<0.05);different concentrations of human recombinated A-SAA with different times did not alter the protein expressions of TNF-a、IL-6 and adiponectin(P﹥0.05)in conditioned media. A-SAA appeared to activate the kinases of JNK and ERK.Conclusions:A-SAA induced the mRNA expression of IL-6 in a dose-dependent manner, the mRNA expression of adiponectin was significantly decreased after stimulating with 10μg/ml A-SAA for 24h, meanwhile the mRNA expressions of TNF-a and IL-6 was significantly increased when stimulated with 10μg/ml A-SAA for 24h. Therefore,A-SAA did not induce insulin resistance of 3T3-L1 adipocytes through increasing the expression of IL-6 and TNF-a and/or decreasing the expression of adiponectin in this study , A-SAA seemed to participate in the insulin resistance of adioicytes through MAPK pathway.更多还原