骨保护素调控肝脏甘油三酯代谢的机制研究

发布时间：2018-05-13 03:13

本文选题：骨保护素 + 非酒精性脂肪肝　；参考：《重庆医科大学》2017年博士论文

【摘要】：第一部分OPG在NAFLD动物模型和病人中的表达目的:探索OPG在NAFLD动物模型和NAFLD病人中的变化,初步了解OPG与NAFLD之间的关系。方法:采用定量PCR法检测c57普食喂养雄性小鼠(20周龄)、HFD小鼠(8周龄开始喂养高脂12周)、ob/ob(普食喂养,20周龄)、db/db(普食喂养,20周龄)、脂联素基因敲除小鼠,NAFLD病人肝脏中OPG m RNA的表达情况。通过western blot方法检测c57、HFD、ob/ob、db/db,NAFLD病人肝脏中OPG蛋白表达情况。免疫组化分析NAFLD病人和正常人肝脏中OPG蛋白的表达。利用不同浓度的葡萄糖和FFAs刺激L02细胞后,测定OPG m RNA的变化。应用定量PCR方法测定不同喂养状态下c57小鼠肝脏中OPG m RNA的表达。结果:在NAFLD动物模型和NAFLD病人中,OPG m RNA和蛋白含量均显著降低(P0.05),葡萄糖可以呈剂量依赖性的刺激OPG m RNA的表达,而FFAs则抑制OPG m RNA的表达。在空腹时,OPG m RNA表达升高,再喂养后OPG m RNA表达降低。结论:OPG与NAFLD的发生发展密切相关。第二部分体外改变OPG表达影响肝细胞甘油三酯的含量目的:体外证实OPG对肝细胞甘油三酯的影响。方法:通过构建OPG过表达或抑制腺病毒,感染L02细胞后,利用FFAs诱导肝细胞脂肪变性。通过油红O染色观察L02细胞脂滴变化情况,甘油三酯测定试剂盒测定肝细胞中TG的含量,PCR法测定甘油三酯代谢相关基因FATP2、FATP4、FATP5、CD36、SREBP1c、FAS、ACC、SCD-1、ch REBP、PPARα、Cpt-1a、Mcad、MTTP、LXR、PXR、RXR、PPARγ等m RNA的变化,western blot法测定甘油三酯代谢相关基因SREBP1c、ACC、FAS、CD36、PPARγ等蛋白的变化,western blot法筛选PKA、Akt、JNK、P38、ERK等信号通路的变化。结果:成功构建OPG过表达和抑制腺病毒。与对照组相比较,OPG过表达组肝细胞脂滴的数量及甘油三酯的含量显著升高(P0.05);且脂肪酸摄取相关基因CD36显著升高,而脂肪酸合成相关基因SREBP1c、FAS、ACC、SCD-1,脂肪酸β氧化相关基因PPARα、Cpt-1a、Mcad,VLDL分泌相关基因MTTP等无明显变化。其次,OPG过表达组核受体基因PPARγ表达明显升高,而p-ERK的活性降低。然而,与对照组比较,OPG表达降低则会抵抗FFAs所诱导的肝细胞脂肪聚集,肝细胞中TG含量降低。脂肪酸摄取相关基因CD36显著降低,而脂肪酸合成相关基因SREBP1c、FAS、ACC、SCD-1,脂肪酸β氧化相关基因PPARα、Cpt-1a、Mcad,VLDL分泌相关基因MTTP等变化不明显。OPG抑制降低了核受体基因PPARγ的表达,增加了p-ERK的活性。结论:体外,OPG促进肝细胞甘油三酯聚集,这一过程可能与CD36和PPARγ的表达密切相关。第三部分OPG基因敲除小鼠抵抗高脂饮食诱导的肝脏脂肪变性目的:通过研究OPG基因敲除小鼠,在体内明确OPG调控肝脏甘油三酯代谢的机制。方法:以杂合子与杂合子配种的方式大量繁殖OPG基因敲除小鼠,获得足够雄性野生型和纯合子小鼠后,在8周龄开始高脂喂养12周,分成WT-SD、OPG-/--SD、WT-HFD、OPG-/--HFD组。每周测定各组小鼠的体重、摄食等,在20周龄时行GTT、ITT等实验,通过眼球取血测定血清生化指标,取肝脏做组织病理切片,油红O染色观察肝脏脂滴多少,HE染色观察肝细胞脂肪变性情况,试剂盒测定肝脏甘油三酯等含量。提取肝脏组织RNA和蛋白做基因表达检测,PCR法测定甘油三酯代谢相关基因FATP2、FATP4、FATP5、CD36、SREBP1c、FAS、ACC、SCD-1、ch REBP、PPARα、Cpt-1a、Mcad、MTTP、LXR、RXR、PXR、FXR、PPARγ等m RNA的变化,western blot法测定甘油三酯代谢相关基因PPARγ、CD36、p-ERK等蛋白的变化。结果:OPG基因敲除小鼠体重明显低于WT小鼠,而摄食无明显变化。GTT、ITT实验显示OPG基因敲除小鼠能抵抗高脂诱导的胰岛素抵抗。血清学检测显示,高脂喂养之后,AST、ALT、TG、TC、FFA、GLU等明显升高,而OPG基因敲除组则低于WT对照组(P0.05)。肝脏组织切片显示高脂喂养后,小鼠肝脏中脂滴明显增多,HE染色结果显示小鼠肝脏发生明显脂肪变性、空泡显著增多,但是OPG-/--HFD明显好于WT-HFD组。OPG-/-组肝脏甘油三酯含量低于WT组。OPG基因敲除组肝细胞脂肪酸摄取相关基因CD36、FATP2、FATP5表达明显降低,而脂肪酸合成相关基因SREBP1c、FAS、ACC、SCD-1,脂肪酸β氧化相关基因PPARα、Cpt-1a、Mcad,VLDL分泌相关基因MTTP等无明显变化。OPG-/-组小鼠肝脏PPARγ表达降低,而p-ERK活性增强。结论:体内,OPG基因敲除小鼠能够明显抵抗高脂饮食喂养诱导的肝脏脂肪变性,可能与脂肪酸摄取相关基因的表达变化密切相关。第四部分OPG调控甘油三酯代谢的机制目的:探索OPG调控肝脏甘油三酯代谢的分子机制。方法:首先利用原代肝细胞分离术,验证OPG过表达对c57原代肝细胞中TG及脂肪酸摄取相关基因CD36,核受体基因PPARγ,p-ERK等的影响。然后分离培养OPG-/-小鼠原代肝细胞,分析其与WT对照组中肝细胞内TG含量及脂肪酸摄取相关基因CD36,核受体基因PPARγ,p-ERK等的影响。进一步,利用CD36-/-原代肝细胞培养,验证OPG是否通过CD36来调控肝细胞中TG的含量。最后通过使用PPARγ抑制剂GW9662、ERK抑制剂SCH772984验证OPG是否通过p-ERK、PPARγ调控CD36的表达。结果:c57原代肝细胞经OPG-Fc处理之后,细胞内TG含量增多,脂肪酸摄取基因CD36表达增加,核受体基因PPARγ表达升高,而p-ERK活性降低。OPG-/-原代肝细胞较WT对照组,细胞内TG减少,脂肪酸摄取基因CD36表达降低,核受体基因PPARγ表达下降,而p-ERK活性增强。在CD36-/-原代中,OPG-Fc则不能增加细胞内TG含量。应用PPARγ抑制剂GW9662后,OPG-Fc不能增加CD36的表达。预先处理ERK抑制剂SCH772984后,OPG-Fc不能增加PPARγ、CD36的表达。结论:OPG通过ERK-PPARγ-CD36轴调控肝脏甘油三酯的含量。
[Abstract]:Part one OPG expression in NAFLD animal models and patients: To explore the changes in OPG in NAFLD animal models and NAFLD patients and to preliminarily understand the relationship between OPG and NAFLD. Methods: the quantitative PCR method was used to detect the male mice (20 weeks old) of C57 universal food (20 weeks old), HFD mice (8 weeks of age to feed high fat 12 weeks), ob/ob (general feeding, 20 weeks old), D B/db (feeding, 20 weeks old), the expression of OPG m RNA in the liver of NAFLD patients with adiponectin gene knockout mice. The expression of C57, HFD, ob/ob, db/db, NAFLD patients' liver was detected by Western blot method. The changes of OPG m RNA were measured after the stimulation of L02 cells. The expression of OPG m RNA in the liver of C57 mice under different feeding States was measured by quantitative PCR. Expression of PG m RNA. The expression of OPG m RNA increased at the empty stomach and OPG m RNA expression decreased after refeeding. Conclusion: OPG and NAFLD are closely related. The second part changes OPG expression in vitro to influence the content of triglycerides in liver cells: in vitro, the effect on hepatocyte glycerol three ester. Method: through construction of overexpression or inhibition Adenovirus, infected with L02 cells, used FFAs to induce fatty degeneration of liver cells. The changes of lipid droplets in L02 cells were observed by oil red O staining. The content of TG in hepatocytes was determined by triglyceride determination kit. PCR method was used to determine triglyceride metabolism related genes FATP2, FATP4, FATP5, CD36, SREBP1c, FAS. PXR, RXR, PPAR gamma and other m RNA changes. Western blot method was used to determine the changes in triglyceride metabolism related genes SREBP1c, ACC, FAS, CD36, PPAR gamma and other proteins. The content of the number and triglyceride increased significantly (P0.05), and the fatty acid uptake related gene CD36 increased significantly, while the fatty acid synthesis related genes SREBP1c, FAS, ACC, SCD-1, fatty acid beta oxidation related genes PPAR a, Cpt-1a, Mcad, VLDL secreted related genes were not significantly changed. Secondly, the gene expression of nuclear receptor gene in the OPG overexpressed group was expressed clearly. Compared with the control group, the decrease of OPG expression resisted the liver cell fat aggregation induced by FFAs, the decrease of TG content in the hepatocytes and the decrease of the fatty acid uptake related gene CD36, and the fatty acid synthesis related genes SREBP1c, FAS, ACC, SCD-1, and fatty acid beta oxidation related genes PPAR a, Cpt-1a, Mcad, FFAs, compared with the control group. The changes of L secretory related gene MTTP do not obviously decrease the expression of PPAR gamma and increase the activity of p-ERK. Conclusion: in vitro, OPG promotes triglyceride aggregation in liver cells, this process may be closely related to the expression of CD36 and PPAR gamma. The third part of OPG knockout mice resists the liver fatty changes induced by high fat diet. Objective: To investigate the mechanism of OPG regulation of triglyceride metabolism in the liver by studying OPG knockout mice. Methods: multiply OPG knockout mice with heterozygotes and heterozygotes, and get enough male wild type and homozygote mice for 12 weeks at 8 weeks of age, and are divided into WT-SD, OPG-/--SD, WT-HFD, OPG-/. Group --HFD. Every week, the weight of mice in each group was measured, feeding and so on. At the age of 20 weeks, GTT, ITT and other experiments were carried out. The serum biochemical indexes were measured by taking blood from the eyeball, taking the liver to make histopathological sections, using the oil red O staining to observe the liver lipid droplets, the HE staining to observe the liver cell fatty degeneration, the determination of liver triglyceride content by the kit and the extraction of liver group. RNA and protein were detected by gene expression, and the changes of triglyceride metabolism related genes such as FATP2, FATP4, FATP5, CD36, SREBP1c, FAS, ACC, SCD-1, CH REBP were measured by PCR. The weight of the knockout mice was significantly lower than that of the WT mice, while the feeding was not significantly changed by.GTT. The ITT test showed that the OPG gene knockout mice could resist high fat induced insulin resistance. The serological test showed that after high fat feeding, AST, ALT, TG, TC, FFA, GLU, and so on were significantly higher, while the OPG gene knockout group was lower than that of the WT control group. The liver tissue section showed high After fat feeding, the lipid droplets in the liver of mice increased obviously. The results of HE staining showed that the liver of mice had obvious fatty degeneration and vacuoles significantly increased, but the content of triglyceride in the liver of group.OPG-/- was better than that of group.OPG-/- in group WT-HFD, and the expression of fatty acid related genes CD36, FATP2 and FATP5 decreased significantly in the group of.OPG gene knockout group WT. Fatty acid synthesis related genes SREBP1c, FAS, ACC, SCD-1, fatty acid beta oxidation related gene PPAR alpha, Cpt-1a, Mcad, VLDL secretion related gene MTTP and so on, the expression of PPAR gamma in the liver of.OPG-/- mice decreased, but p-ERK activity was enhanced. Sex may be closely related to the changes in the expression of fatty acid uptake related genes. Fourth the mechanism of regulating triglyceride metabolism by OPG: To explore the molecular mechanism of OPG regulation of triglyceride metabolism in the liver. Methods: first of all, primary hepatocyte isolation was used to verify the TG and fatty acid uptake related gene CD3 in C57 primary hepatocytes by OPG overexpression. 6, the effect of nuclear receptor gene PPAR gamma, p-ERK and so on. Then, the primary hepatocytes of OPG-/- mice were isolated and cultured, and the effects of TG content and fatty acid uptake related gene CD36, nuclear receptor gene PPAR gamma, p-ERK and so on in the liver cells of the WT control group were analyzed. Further, using CD36-/- primary hepatocyte culture to verify whether OPG was regulated by CD36 to regulate the liver cells. Finally, the expression of CD36 is regulated by the use of PPAR gamma inhibitor GW9662 and ERK inhibitor SCH772984 to verify whether OPG passes through p-ERK and PPAR gamma regulates the expression of CD36. Results: after OPG-Fc treatment, C57 primary hepatocytes are increased, the expression of fatty acid uptake gene is increased, the expression of fatty acid uptake gene is increased, and the expression of nuclear receptor gene is increased, and the activity of PPAR is reduced. Compared with the WT control group, the intracellular TG decreased, the expression of fatty acid uptake gene CD36 decreased, the PPAR gamma expression of the nuclear receptor gene decreased and the p-ERK activity increased. In the original CD36-/- generation, OPG-Fc could not increase the intracellular TG content. After GW9662, OPG-Fc could not increase the expression of CD36. OPG-Fc can not increase the expression of PPAR gamma and CD36. Conclusion: OPG regulates the triglyceride level of liver by ERK-PPAR -CD36 -CD36 axis.

【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R575.5

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