1型多聚ADP核糖合成酶通过PPARα通路抑制脂肪酸氧化导致脂质代谢紊乱的机制研究

发布时间：2018-05-03 04:24

本文选题：1型多聚ADP核糖合成 + 过氧化物酶体增殖物受体α　；参考：《华中科技大学》2014年博士论文

【摘要】：第一部分1型多聚ADP核糖合成酶在高饱和脂肪酸饮食所致甘油三酯代谢紊乱过程中的作用机制研究目的：使用1型多聚ADP核糖合成酶(poly(ADP-ribose) polymerase1,PARP1)基因敲除小鼠,通过给予高饱和脂肪酸饮食喂养构建高甘油三酯血症动物模型,探讨PARP1在高甘油三酯血症中的作用和机制。方法：选择体重在18-20g,8-10周龄的雄性野生型129S1/SvImJ小鼠及PARP1基因敲除小鼠(PARP1-/-,129S-Parpltmlzqw/J,PKO)为实验对象,各随机分为4组,分别给予普通饮食和高饱和脂肪酸饮食喂养。在实验开始第0周,首次测量体重,并经内眦静脉采血,分离血清后检测血甘油三酯和非酯化脂肪酸含量。以后于第14、28天测量一次小鼠体重。在实验第27天,小鼠禁食6小时后,分别行小鼠灌胃给予葡萄糖耐量试验、腹腔注射胰岛素耐量试验,评价小鼠胰岛素抵抗状态。第28天小鼠在禁食8小时后,取血分离血清,再次检测血甘油三酯和非酯化脂肪酸含量。处死小鼠后,取肝脏织样本使用多聚甲醛固定留作病理切片,或者冻存作分子生物学检测。病理学检测包括HE及油红染色肝脏组织切片观察肝脏脂肪变性情况；使用蛋白印迹实验等方法检测老鼠肝脏中PARP活性,real time RT-PCR检测肝脏组织中甘油三酯代谢相关基因RNA表达水平,凝胶迁移电泳检测肝脏组织中过氧化物酶体增殖物受体a的DNA结合能力。结果：高饱和脂肪酸饮食喂养后,小鼠肝脏组织中PARP活性明显增高。PARP1敲除或PARP1抑制剂均可以明显改善高饱和脂肪酸饮食所致的血脂异常和肝脏脂质沉积。敲除PARP1或PARP抑制剂可以增强过氧化物酶体增殖物受体α的DNA结合能力,并显著增加参与脂肪酸氧化相关基因的表达水平。结论：PARP1基因敲除后可以明显改善由高饱和脂肪酸饮食诱导的甘油三酯代谢紊乱,PARP1在脂质代谢紊乱过程中发挥重要作用。第二部分不同种类脂肪酸对1型多聚ADP核糖合成酶活性作用的研究目的：1型多聚ADP核糖合成酶(PARP1)的过度激活在各种生理病理过程中发挥着关键的作用,研究不同种类脂肪酸对PARP1活性的影响,探索高饱和脂脂酸饮食引起脂质代谢紊乱的原因。方法：培养野生型小鼠肝脏原代细胞,使用相同浓度的单不饱和脂肪酸、多不饱和脂肪酸和饱和脂肪酸刺激肝脏细胞,检测肝脏细胞中PARP1的活性。并且使用免疫共沉淀观察不同种类脂肪酸刺激细胞后过氧化物酶体增殖物受体α(PPARa)的多聚ADP核糖化水平。同时培养野生型小鼠和PARP1基因敲除小鼠肝脏原代细胞,观察不同种类脂肪酸对脂肪酸代谢相关基因表达的影响。结果：单不饱和脂肪酸与多不饱和脂肪酸不能使PARP1活性的增加,而饱和脂肪酸则可以激活PARP1,且呈浓度依赖性。并且较之单不饱和脂肪酸与多不饱和脂肪酸,饱和脂肪酸增加PPARα的多聚ADP核糖化水平。实时定量PCR结果表明饱和脂肪酸在野生型细胞中不能够增加PPARα下游靶基因的表达,但是在PARP1敲除细胞中则可以上调PPARα下游靶基因的表达。结论：三种脂肪酸中只有饱和脂肪酸可以激活PARP1,饱和脂肪酸在PARP1敲除细胞中可以增加PPARα下游靶基因的表达,PARP1的激活是高饱和脂肪酸饮食引起脂质代谢紊乱的关键因素。第三部分人肝脏组织1型多聚ADP核糖合成酶活性的观察目的：观察人肝脏组织中1型多聚ADP核糖合成酶(PARP1)活性以及与肝脏组织中脂质含量的关系。方法：收集临床病人的肝脏组织,选取病理学检查正常的肝脏组织作为实验对象,使用Universal colorimetric PARP assay试剂盒检测肝脏组织标本中PARP活性,并且检测相应肝脏组织中甘油三酯和非酯化脂肪酸的含量,使用Pearson相关模型分析PARP活性与脂质含量之间的关系。并且用PARP1激动剂和抑制剂孵育人肝癌细胞系hepG2细胞,用油红O检测脂质沉积程度。结果：人肝脏组织中PARP活性与甘油三酯(r=0.509,p0.001)和非酯化脂肪酸(r=0.454,p=0.002)的含量呈正相关,PARP1激动剂可以增加hepG2细胞中的脂质沉积,反之,PARP1抑制剂可以减少hepG2中细胞的脂质沉积。结论：PARP1在人肝脏甘油三酯沉积中起着非常重要的作用。
[Abstract]:Study on the Mechanism of First Part 1 Poly ( ADP - ribose ) Synthetase ( ADP - ribose ) Synthetase in the Metabolic Disorder of Triglyceride Caused by Hypersaturated Fatty Acid Diet

Objective : To study the role and mechanism of PARP1 in hypertriglycerides by knockout mice with poly ( ADP - ribose ) polymerase1 ( PARP1 ) gene knockout mice .

Methods : Male wild - type 129S1 / SvImJ mice and PARP1 knockout mice ( PARP1 - / - , 129S - Parpltmlzqw / J , Kunming ) were randomly divided into 4 groups .
The mRNA expression level of triglyceride - related genes in liver tissues was detected by Western blot and real time RT - PCR . The DNA binding ability was detected by gel migration electrophoresis .

Results : After feeding with high - saturated fatty acid , the activity of parP1 knock - out or PARP1 inhibitor can obviously improve the abnormal blood lipid and lipid deposition caused by high - saturated fatty acid diet . The knock - out PARP1 or parp inhibitor can enhance the DNA binding ability of the peroxidase complex receptor 伪 and increase the expression level of the related genes involved in fatty acid oxidation .

Conclusion : PARP1 gene knockout can obviously improve triglyceride metabolism induced by high saturated fatty acid diet , and PARP1 plays an important role in lipid metabolism disorder .

Effect of Different Fatty Acids on Activity of Poly ( ADP - ribose ) Synthetase in Part Two

Objective : To investigate the effect of different kinds of fatty acids on PARP1 activity and to explore the causes of lipid metabolism disorder caused by high saturated fatty acid diet .

Methods : The primary cells of wild type mouse liver were cultured . Single unsaturated fatty acids , polyunsaturated fatty acids and saturated fatty acids of the same concentration were used to stimulate liver cells to detect the activity of PARP1 in liver cells .

Results : Monounsaturated fatty acids and polyunsaturated fatty acids could not increase PARP1 activity , while saturated fatty acids could activate PARP1 and increase PPAR伪 ' s poly ADP - ribose level . The real - time quantitative PCR results showed that saturated fatty acids could not increase the expression of PPAR伪 downstream target genes in wild - type cells , but could up - regulate the expression of PPAR伪 downstream target genes in PARP1 knockout cells .

Conclusion : Only saturated fatty acids in three fatty acids can activate PARP1 , and saturated fatty acids can increase the expression of PPAR伪 downstream target gene in PARP1 knockout cells . PARP1 activation is a key factor in lipid metabolism disorder caused by high saturated fatty acid diet .

the activity of poly ADP - ribose synthase in human liver tissue of the third part

Objective : To observe the activity of poly ADP - ribose synthase ( PARP1 ) in human liver tissue and its relationship with lipid content in liver tissue .

Methods : The liver tissues of clinical patients were collected , and the normal liver tissues were selected as experimental subjects . The content of triglyceride and non - esterified fatty acids in liver tissues was detected by using the Universal colorimetric method . The relationship between the activity and lipid content of liver tissues was analyzed by Pearson correlation model , and the degree of lipid deposition was detected by using the PARP1 agonist and inhibitor to incubate the hepG2 cells of human hepatoma cell line hepG2 .

Results : In human liver tissue , the activity of PARP1 was positively correlated with triglyceride ( r = 0.509 , p0.001 ) and non - esterified fatty acid ( r = 0.454 , p = 0.002 ) . The PARP1 agonist could increase the lipid deposition in hepG2 cells , whereas PARP1 inhibitors could reduce the lipid deposition of cells in hepG2 .

Conclusion : PARP1 plays a very important role in the deposition of triglyceride in human liver .

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R575.5

【共引文献】