【作者】 臧丽；

【导师】 母义明；

【作者基本信息】 中国人民解放军军医进修学院 ， 内科学， 2010， 博士

【摘要】 目的：探讨人白血病相关蛋白16 (Leukemia Related Protein 16, LRP16)对3T3-L1脂肪细胞、C2-C12成肌细胞、HepG2肝癌细胞胰岛素抵抗的影响及可能的分子机制。方法：(1)利用脂质体转染及慢病毒介导的小干扰RNA(small interference RNA, siRNA)技术构建过表达LRP16细胞系、抑制表达LRP16细胞系及对照细胞系。(2)将3T3-L1前脂肪细胞常规诱导成熟,倒置显微镜观察及油红染色判断过表达及抑制表达LRP16对3T3-L1前脂肪细胞分化的影响；Q-PCR (Real-time Quantitative PCR)法检测过表达及抑制表达LRP16对3T3-L1脂肪细胞分化相关因子CAAT增强子结合蛋白α(CAAT/Enhancer Binding Protein alpha, C/EBPα)、过氧化物酶体增殖物激活受体γ(Peroxisome Proliferator activated receptor gamma, PPARγ),以及肿瘤坏死因子α(Tumor Necrosis Factor alpha, TNFα)、白介素(Interleukin, IL)-6、抵抗素(Resistin)、脂联素(Adiponectin) mRNA表达的影响。(3) 2-deoxy-[3H]-D-glucose检测过表达及抑制表达LRP16对3T3-L1脂肪细胞、C2-C12成肌细胞、HepG2肝癌细胞胰岛素刺激状态下葡萄糖摄取率的影响；Western Blot法检测上述细胞中过表达及抑制表达LRP16对胰岛素受体底物(Insulin Receptor Substrate, IRS)-1信号通路关键蛋白pIRS-1(Ser307)、pIRS-1(Tyr)、PI3-K(p85)、pAkt(Ser473)、pAkt(Thr308)表达的影响。(4)将pCMX-PPARy(pCMX-PPARα)、pGL3-PPRE、pcDNA-3.1、pcDNA-3.1-16、pRL-TK共转染COS-7细胞,双荧光素酶报告检测系统检测过氧化物酶体增生因子反应元件(Peroxisome Proliferator Response Element, PPRE)相对荧光素酶活性以反映PPARγ及过氧化物酶体增殖物激活受体α(Peroxisome Proliferator activated receptor alpha, PPARa)的转录活性；Western Blot法检测过表达及抑制表达LRP16对3T3-L1脂肪细胞、C2-C12成肌细胞PPARγ、葡萄糖转运蛋白(Glucose Transporter, GLUT)-4表达的影响,及对HepG2肝癌细胞PPARα、脂蛋白脂酶(Lipoprotein Lipase, LPL)蛋白表达的影响。结果：(1)成功构建了过表达及抑制表达LRP16的3T3-L1、C2-C12、HepG2细胞系与对照细胞系。(2)过表达LRP16的脂肪细胞脂滴大而少,分散而不均匀,呈明显指环样的脂滴较少,油红O染色示570nm OD值低于对照细胞；抑制表达LRP16的脂肪细胞脂滴小而多,密集而均匀,整个视野呈“油菜花样”改变,油红O染色示570nm OD值高于对照细胞。过表达LRP16的脂肪细胞C/EBPα、PPARγ及脂联素mRNA表达低于对照细胞,TNFα、IL-6、抵抗素的mRNA表达高于对照细胞；抑制表达LRP16的脂肪细胞C/EBPα、PPARyγ及脂联素mRNA表达高于对照细胞,TNFα、IL-6、抵抗素的mRNA表达低于对照细胞。(3)过表达LRP16抑制3T3-L1脂肪细胞、C2-C12成肌细胞及HepG2肝癌细胞胰岛素刺激的葡萄糖摄取,抑制表达LRP16则增加3T3-L1脂肪细胞、C2-C12成肌细胞及HepG2肝癌细胞胰岛素刺激的葡萄糖摄取。过表达LRP16上调三种细胞pIRS-1(Ser307)蛋白的表达,下调pIRS-1(Tyr)、PI3-K(p85)、pAkt(Ser473)、pAkt(Thr308)蛋白的表达,相反,抑制表达LRP16则下调三种细胞pIRS-1(Ser307)蛋白的表达,上调pIRS-1(Tyr)、PI3-K(p85)、pAkt(Ser473)、pAkt(Thr308)蛋白的表达。(4)LRP16剂量依赖性的降低PPARγ的转录活性,当pcDNA3.1-16量为0.4μg时,pGL3-PPRE的相对荧光素酶活性降低为对照组的43%(P<0.01),当pcDNA3.1-16量为0.5gg时,pGL3-PPRE的相对荧光素酶活性降低为对照组的27%(P<0.01)；LRP16剂量依赖性的降低PPARa的转录活性：当pcDNA3.1-16量为0.4μg时,pGL3-PPRE的相对荧光素酶活性降低为对照组的38%(P<0.01),当pcDNA3.1-16量为0.5μg时,pGL3-PPRE的相对荧光素酶活性降低为对照组的35%(P<0.01)。过表达LRP16下调3T3-L1脂肪细胞、C2-C12成肌细胞PPARy、GLUT-4蛋白的表达,抑制表达LRP16则上调3T3-L1脂肪细胞PPARy、GLUT-4蛋白的表达；过表达LRP16下调HepG2肝癌细胞PPARa、LPL蛋白的表达。结论：(1)我们成功构建了过表达及抑制表达LRP16的3T3-L1、C2-C12、HepG2细胞系与对照细胞系,为后续实验提供了细胞模型。(2)LRP16通过抑制脂肪细胞分化,影响IRS-1信号通路,抑制PPARy及PPARa的转录活性等多条途径导致外周组织胰岛素抵抗。更多还原

【Abstract】 Objective:To investigate the effect of LRP16 on insulin resistance in 3T3-L1 adipocyte, C2-C12 sarcoblast and HepG2 hepatoma carcinoma cell and the possible molecular mechanisms.Methods:(1) Lipidosome transfection and lentivirus mediated siRNA technology were used to construct the cell lines with over-expression of LRP16, down-expression of LRP16 and the conrtol cell lines. (2) 3T3-L1 preadipocytes were differentiated using the regular methods, the effect of LRP16 on differentiation of 3T3-L1 preadipocytes were detected using inverted microscope and red oil staining; The effect of LRP16 on mRNA expression of C/EBPα, PPARy, TNFa, IL-6, Resistin and Adiponectin were measured using Quantitative Real-time PCR. (3) 2-deoxy-[3H]-D-glucose was used to detect the effect of LRP16 on insulin stimulated glucose uptake in 3T3-L1 adipocyte, C2-C12 sarcoblast and HepG2 hepatoma carcinoma cell; Effect of LRP16 on the expression of key protein in IRS-1 signaling pathway, such as pIRS-1(Ser307)、pIRS-1(Tyr)、PI3-K(p85)、pAkt(Ser473)、pAkt(Thr308), was detected using Western Blot. (4) The strucural expression vectors, including pCMX-PPARy (pCMX-PPARa), pGL3-PPRE, pcDNA-3.1, pcDNA-3.1-16 and the internal conrtol vector pRL-TK were transiently co-transfected into COS-7 cells, then the PPRE relative luciferase activity which reflected the transcriptional activity of PPARy and PPARa was detected using Dual-luciferase Reporter Gene Assay system. Western Blot was used to detect the effect of LRP16 on the expression of PPARy and GLUT-4 in 3T3-L1 adipocyte, C2-C12 sarcoblast, and the expression of PPARa, LPL in HepG2 hepatoma carcinoma cell.Results:(1) We constructed the cell lines with over-expression of LRP16, down-expression of LRP16 and the conrtol cell lines in 3T3-L1 adipocyte, C2-C12 sarcoblast and HepG2 hepatoma carcinoma cell successfully. (2) The cytolipin in 3T3-L1 adipocyte with over-expression of LRP16 was large, few, uneven, the red oil staining showed that the value of OD in 570nm was lower than the control cells; On the contrary, the cytolipin in 3T3-L1 adipocyte with down-expression of LRP16 was small, intensive and even, the whole visual fields was cauliflower-like, the red oil staining showed that the value of OD in 570nm was higher than the control cells. The mRNA expression of C/EBPa, PPARy and Adiponectin in cells with over-expression of LRP16 was lower than the control cells, but the mRNA expression of TNFα, IL-6, Resistin was higher than the control cells. On the contrary, when the LRP16 was down-expressed, the results were just on the opposite. (3) Over-expression of LRP16 decreased the insulin stimulated glucose uptake in 3T3-L1 adipocyte, C2-C12 sarcoblast and HepG2 hepatoma carcinoma cell, but, down-expression of LRP16 increased the insulin stimulated glucose uptake in these cells. Protein expression of pIRS-1(Ser307) was increased when LRP16 was over-expressed, but, the expression of pIRS-1(Tyr). PI3-K(p85)、pAkt(Ser473)、pAkt(Thr308) were decreased. On the contrary, when the LRP16 was down-expressed, the results were just on the opposite. (4) LRP16 decreased the transcriptional activity of PPARy in a dose dependent manner, the relative luciferase activity of PPRE accounted for 38%(P<0.01) of the control group at pcDNA3.1-16 0.4μg, and 35%(P<0.01) of the control group at pcDNA3.1-16 0.5μg. LRP16 decreased the transcriptional activity of PPARa in a dose dependent manner, the relative luciferase activity of PPRE accounted for 43%(P<0.01) of the control group at pcDNA3.1-16 0.4μg, and 27%(P<0.01) of the control group at pcDNA3.1-16 0.5μg. overexpression of LRP16 decreased the protein expression of PPARy, GLUT-4 in 3T3-L1 adipocyte, C2-C12 sarcoblast, and the protein expression of PPARa、LPL in HepG2 hepatoma carcinoma cell, but, when LRP16 was down-expressed, the results were on the opposite.Conclusions:(1) We successfully constructed the cell lines with over-expression of LRP16, down-expression of LRP16 and control cell lines, which provided cell model for the follow up experiment. (2) LRP16 caused insulin resistance through several pathways, including inhibiting the differentiation of adipocytes, influencing IRS-1 signaling pathway and inhibiting the transcriptional activity of PPARy and PPARa.更多还原