微小RNA在高血糖“代谢记忆”中的作用及机制研究

发布时间：2018-04-18 23:22

本文选题：高血糖 + 代谢记忆　；参考：《华中科技大学》2015年博士论文

【摘要】：目的：建立高血糖“代谢记忆”的动物及细胞模型,观察“代谢记忆”状态下大鼠代谢指标、NF-κB(核转录因子-κB)p65亚单位及前炎症因子的表达变化。方法： 1. Wistar大鼠分为3组：(1)正常对照组,注射柠檬酸钠缓冲液：(2)代谢记忆组,注射链脲佐菌素(STZ)成模后维持高血糖水平12周,从第13周开始皮下注射胰岛素控制血糖水平在正常范围；(3)持续高糖组,注射STZ成模后维持24周高血糖状态。 2.人主动脉内皮细胞(HAECs)分为4组：(1)正糖组,含5.5mM葡萄糖的培养基培养7天；(2)渗透压对照组,含30mM甘露醇的培养基培养7天；(3)代谢记忆组,含30mM葡萄糖的培养基培养1天后,改为含5.5mM葡萄糖的培养基培养6天；(4)高糖组,含30mM葡萄糖的培养基培养7天。 3.采用实时定量聚合酶链反应(PCR)检测各组大鼠主动脉组织及细胞样本中p65、单核细胞趋化因子-1(MCP-1)和白介素-6(IL-6)的mRNA表达水平：采用免疫印迹(Western blot)法检测p65蛋白表达水平。结果： 1.正常对照组大鼠体重持续增加,代谢记忆组及持续高糖组大鼠在注射STZ后体重增加速度均低于正常对照组；代谢记忆组大鼠体重增速高于持续高糖组,低于正常对照组。 2.代谢记忆组大鼠主动脉组织及细胞样本中p65、MCP-1及IL-6的表达水平上升。结论： 1.成功建立高血糖“代谢记忆”的动物及细胞模型。 2.高血糖“代谢记忆”中p65及前炎症因子MCP-1、IL-6升高,NF-κB通路持续激活。目的：分别观察miR-146a和miR-484在高血糖“代谢记忆”状态下的表达变化,通过生物信息学方法寻找并确定其靶基因,观察靶基因在高血糖“代谢记忆”状态下的表达变化。方法： 1.采用茎环荧光实时定量PCR法检测各组大鼠主动脉组织及细胞样本中miR-146a和miR-484表达水平变化。 2.使用生物信息学方法寻找niR-146a和miR-484可能的靶基因；检测各组动物及细胞样本中靶基因的表达。结果： 1.代谢记忆组大鼠主动脉组织及细胞样本中miR-146a的表达水平下降。 2.靶基因预测软件预测到miR-146a的靶基因包括白介素-1受体相关激酶1(IRAK1)和肿瘤坏死因子受体相关因子6(TRAF6);代谢记忆组动物及细胞样本中IRAK1、TRAF6的蛋白表达水平上升。 3.代谢记忆组动物主动脉组织及细胞样本中miR-484的表达水平下降。 4.靶基因预测软件预测到miR-484的靶基因是血管内皮生长因子B(VEGFB);代谢记忆组动物主动脉组织及细胞样本中VEGFB的表达水平上升。结论： 1.高血糖“代谢记忆”时miR-146a表达下降,其靶基因IRAK1和TRAF6表达增加。 2.高血糖“代谢记忆”时miR-484表达下降,其靶基因VEGFB表达增加。目的：在前述研究建立的高血糖“代谢记忆”动物及细胞模型的基础上,通过细胞转染探究miR-146a和miR-484对NF-κB p65亚单位及前炎症因子的作用机制。方法： 1.通过转染miR-146a mimics/inhibitors,上调或下调HAECs中miR-146a的表达,检测预测靶基因蛋白表达水平、NF-κB p65亚单位、MCP-1及IL-6的表达水平。 2.通过转染miR-484mimics/inhibitors,上调或下调HAECs中miR-484的表达水平,检测VEGFB、NF-κB p65亚单位、MCP-1及IL-6的表达水平。 3.通过转染VEGFB过表达质粒或VEGFB siRNA,上调或下调HAECs中VEGFB的表达,检测NF-κB P65亚单位、MCP-1及IL-6的表达水平。结果： 1.转染miR-146a mimics后,HAECs中p65、MCP-1及IL-6的表达水平下降；转染了miR-146a inhibitors,细胞内p65、MCP-1及IL-6的表达水平上升。 2.上调miR-146a表达可使IRAK1、TRAF6的表达水平下降;下调miR-146a表达后,IRAK1、TRAF6的表达水平上升。 3.转染miR-484mimics后,HAECs中miR-484表达上升,p65、MCP-1及IL-6的表达水平下降；转染miR-484inhibitors, miR-484表达下降,NF-κB p65亚单位、MCP-1及IL-6的表达水平上升。 4.转染miR-484mimics, HAECs中VEGFB的表达水平下降；转染miR-484inhibitors, VEGFB的表达水平上升。 5.上调HAECs中VEGFB表达,NF-κB p65亚单位、MCP-1及IL-6的表达水平上升；下调VEGFB表达后细胞内p65、MCP-1及IL-6的表达水平下降。结论： 1.高血糖“代谢记忆”时miR-146a表达下降,并可通过其靶基因IRAK1和TRAF6来调节NF-κB p65及前炎症因子的表达。 2.高血糖“代谢记忆”时miR-484表达持续下降,并可通过其靶基因VEGFB调节p65及前炎症因子的表达。
[Abstract]:Objective: to establish animal model of hyperglycemia and cellular metabolic memory, metabolism of rats. The "metabolic memory" state, NF- kappa B (NF kappa B) expression of p65 subunit and proinflammatory cytokines.
Method:
1. Wistar rats were divided into 3 groups: (1) normal control group, injection of sodium citrate buffer: (2) the metabolic memory group, injection of streptozotocin (STZ) injection to maintain high blood glucose levels for 12 weeks, beginning of subcutaneous injection of insulin to control blood sugar levels in the normal range (from thirteenth weeks; 3) sustained high glucose group, high glucose for 24 weeks after injection of STZ into modules.
2. human aortic endothelial cells (HAECs) were divided into 4 groups: (1) normal glucose group, medium containing 5.5mM glucose cultured for 7 days; (2) the osmotic pressure of the control group, 30mM medium containing mannitol cultured for 7 days; (3) the metabolic memory group, medium containing 30mM glucose after 1 days of culture instead, a medium containing 5.5mM glucose cultured for 6 days; (4) high glucose group, medium containing 30mM glucose cultured for 7 days.
3. real time quantitative polymerase chain reaction (PCR) was used to detect the expression level of p65, monocyte chemoattractant factor -1 (MCP-1) and interleukin -6 (IL-6) in rat aortic tissue and cell samples. The expression level of p65 protein was detected by immunoblot (Western blot).
Result:
1. the weight of the rats in the normal control group increased continuously, and the weight gain rate of the rats in the metabolic memory group and the continuous high glucose group were lower than those in the normal control group after injection of STZ. The body weight gain of the metabolic memory group was higher than that of the continuous high glucose group, which was lower than that of the normal control group.
The expression levels of p65, MCP-1 and IL-6 in the aortic tissue and cell samples in 2. metabolic memory rats were increased.
Conclusion:
1. the animal and cell model of hyperglycemia "metabolic memory" was successfully established.
2. high blood glucose metabolic memory p65 and proinflammatory cytokines MCP-1, IL-6 increased, NF- sustained activation of B pathway.
Objective: To observe the expression changes of miR-146a and miR-484 in hyperglycemic metabolic memory state, and to find and identify their target genes by bioinformatics, and to observe the expression changes of target genes in hyperglycemic "metabolic memory" state.
Method:
1. the expression of miR-146a and miR-484 in the aorta tissue and cell samples of each group was detected by real time quantitative PCR method with stem ring fluorescence.
2. use bioinformatics to find the possible target genes of niR-146a and miR-484, and detect the expression of target genes in the animal and cell samples of each group.
Result:
The expression level of miR-146a in the aortic tissue and cell samples in the 1. metabolic memory group was decreased.
2. target gene prediction software predicted the target genes of miR-146a including interleukin -1 receptor related kinase 1 (IRAK1) and tumor necrosis factor receptor related factor 6 (TRAF6), and the protein expression level of IRAK1 and TRAF6 in animal and cell samples of metabolic memory group increased.
The expression level of miR-484 in the aortic tissue and cell samples in the 3. metabolic memory group was decreased.
4. target gene prediction software predicted that the target gene of miR-484 was vascular endothelial growth factor B (VEGFB), and the expression level of VEGFB in aortic tissue and cell samples of metabolic memory group increased.
Conclusion:
When 1. hyperglycemia "metabolic memory", the expression of miR-146a decreased, and the expression of target gene IRAK1 and TRAF6 increased.
When 2. hyperglycemia "metabolic memory", the expression of miR-484 decreased and the expression of target gene VEGFB increased.
Objective: based high blood glucose metabolic memory cell and animal model in the research on the inquiry by cell transfection mechanism of miR-146a and miR-484 on NF- kappa B subunit p65 and proinflammatory cytokines.
Method:
1. through transfection of miR-146a mimics/inhibitors, up regulation or down regulation of miR-146a expression in HAECs, detection of target gene protein expression level, NF- kappa B p65 subunit, MCP-1 and IL-6 expression level were detected.
2. through transfection of miR-484mimics/inhibitors, the expression level of miR-484 in HAECs was up or down, and the expression level of VEGFB, NF- kappa B p65 subunit, MCP-1 and IL-6 was detected.
3. through transfection of VEGFB overexpression plasmid or VEGFB siRNA, the expression of VEGFB in HAECs was up-regulated or down regulated, and the expression level of NF- kappa B P65 subunit and MCP-1 and IL-6 was detected.
Result:
1. after transfection of miR-146a mimics, the expression level of p65, MCP-1 and IL-6 decreased in HAECs, and the expression level of p65, MCP-1 and IL-6 increased in transfected miR-146a inhibitors.
2. up - regulation of miR-146a expression could decrease the expression level of IRAK1 and TRAF6, and the expression level of IRAK1 and TRAF6 increased after downregulation of miR-146a.
3. after transfection of miR-484mimics, the expression of miR-484 in HAECs increased, and the expression levels of p65, MCP-1 and IL-6 decreased. The expression of miR-484inhibitors and miR-484 decreased, and the expression level of NF- and B p65 subunits increased.
4. transfected miR-484mimics, the expression level of VEGFB in HAECs decreased, and the expression level of VEGFB was increased by transfection of miR-484inhibitors.
5. upregulated VEGFB expression in HAECs, increased NF- kappa B p65 subunit, MCP-1 and IL-6 expression, and decreased p65 expression, MCP-1 and IL-6 expression after VEGFB expression.
Conclusion:
1. high blood glucose metabolic memory decreased expression of miR-146a, and through the expression of its target gene IRAK1 and TRAF6 to regulate NF- kappa B p65 and proinflammatory cytokines.
2. high blood glucose metabolic memory miR-484 expression continued to decline, and the expression of p65 can be regulated by proinflammatory cytokines and its target gene VEGFB.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R587.1

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