ApoE缺失小鼠炎性因子关键基因的表达研究

发布时间：2018-01-13 15:02

本文关键词：ApoE缺失小鼠炎性因子关键基因的表达研究　出处：《山东师范大学》2008年硕士论文　论文类型：学位论文

【摘要】： 心脑血管疾病正严重威胁着人类的健康,由其导致的死亡率呈逐年上升趋势,动脉粥样硬化(atherosclerosis, AS)是其病理基础。关于AS的发病机制存在多种学说,主要是由多种因素引起的慢性炎症反应学说以及脂代谢紊乱学说,而在机体内慢性炎症反应和脂代谢紊乱相互影响、共同参与并促进了AS发生发展。载脂蛋白E基因缺失(apoE-/-)小鼠是目前常用的研究AS的模型,在普通饮食条件下可自发产生AS,随着小鼠年龄的增长病变逐渐加重,并且其AS病变过程中呈现出的病理特征与人AS非常相似,因此apoE-/-小鼠是研究AS发生机制,揭示AS发生发展机理的优良动物模型。然而在apoE-/-小鼠AS发生早期,病变较轻、干扰因素较少的情况下,较系统的检测炎症相关基因的时序表达差异以及各基因之间的相互作用的报道尚不多见。为此,本实验利用该小鼠为模型,检测AS早期小鼠主动脉、肝脏和脾脏中多种炎症相关基因的时序表达,旨在揭示在AS发生早期起关键作用的危险因子,并初步探讨其作用机理,为进一步深入开展分子生物学研究奠定基础。本实验选用apoE-/-小鼠和具有相同遗传背景(C57BL/6J)的野生型(wild type, WT)小鼠为实验对象,利用半定量RT-PCR以及Real-time RT-PCR技术,检测apoE-/-小鼠在1、2和3月龄3个年龄段主动脉以及14天、1、2和3月龄4个年龄段肝脏和脾脏中炎症相关基因的时序表达特点;并通过血清生化指标的检测结合主动脉根部和肝脏病理形态学特征的分析,探讨炎症相关基因的时序表达与AS早期病变的关系。通过实验得到如下结果: 1.利用半定量RT-PCR在主动脉检测的11条炎症相关基因中,apoE-/-与WT小鼠相比,1、2和3月龄时IL-1β的mRNA表达水平均显著上调。1月龄时VCAM-1、IкB-α、TGF-β和SOD1,2月龄时PDGF-α和CD36,3月龄时TNF-α和MMP2的mRNA表达水平与同龄WT小鼠相比均显著上调,其它年龄段无显著变化;而Albumin在1和2月龄时显著降低。在肝脏检测的7条炎症相关基因中,CRP和JAK1的mRNA表达水平在14天到2月龄时无显著变化,3月龄时显著上调。NF-кB在各年龄段apoE-/-小鼠主动脉和肝脏中的基因表达量与同龄WT小鼠相比均无显著差异。在肝脏中检测的其它3条基因在14天至3月龄期间与同龄的WT小鼠相比并无显著变化。在脾脏中检测的8条炎症相关基因中,apoE-/-与WT小鼠相比,2月龄时GM-CSF和SOD1的mRNA表达水平显著升高,其它均无显著变化。 2.利用Real-time RT-PCR在主动脉检测的8条炎症相关基因中,3月龄时IL-1βmRNA表达水平与同龄WT小鼠相比升高约27倍,TNF-αmRNA在1月龄和3月龄时表达水平与同龄WT小鼠相比分别升高约3倍和2倍,3月龄时MCP-1基因表达水平升高约3倍,1月龄和3月龄时ICAM-1基因表达水平均显著上调约3倍,1月龄和3月龄时VCAM-1基因表达水平均显著上调约23倍和2倍,1月龄和3月龄时GM-CSF基因表达水平与同龄WT小鼠相比均上调,NF-кB和IкB-α基因在1和3月龄时均无显著变化。 3. 14天至3月龄4个年龄段的apoE-/-小鼠血清TC、LDL-C和HDL-C水平均显著高于同龄WT小鼠;TG水平从1月龄开始比同龄WT小鼠显著升高,并随小鼠年龄的增长呈现明显的上升趋势。 4. 1月龄apoE-/-小鼠主动脉内膜出现光镜下可见的轻微的脂质沉积,沉积面积较小,主要分布在主动脉瓣膜附近,随年龄增长病变逐渐加重,到3月龄时脂质沉积面积变大、沉积的数量变多,也不仅仅局限在瓣膜处。 5.肝脏病理形态切片观察发现apoE-/-小鼠从1月龄开始,肝脏细胞内出现光镜下可见的脂肪滴,脂肪滴较小且数量较少,随小鼠年龄增长脂肪滴逐渐变大,数量变多,直至形成空泡,表现出明显的肝细胞脂质堆积和肝脏脂肪性病变。以上实验结果显示,随着小鼠年龄的增长,AS病变逐渐加重,炎症相关基因与WT小鼠相比,出现时序性差异表达,这些时序表达差异的炎症相关基因是NF-κB信号通路的上下游基因,构成了以NF-κB为核心的复杂调控网络,它们之间相互作用、相互影响,共同参与慢性炎症过程,在apoE-/-小鼠AS的早期发生发展中发挥重要作用。
[Abstract]:Cardiovascular disease is a serious threat to human health, the resulting mortality increased year by year, atherosclerosis (atherosclerosis, AS) is the pathological basis. The pathogenesis of AS has many kinds of theories, is mainly caused by a variety of factors of chronic inflammation and lipid metabolism in the body theory, and chronic inflammation the reaction and lipid metabolism disorders affect each other, participate in and promote the development of AS.
Apolipoprotein E gene deficient (apoE-/-) mice are currently on a common AS model, in the ordinary diet under the condition of spontaneous AS with aging changes gradually aggravated, and presents the AS of the pathological changes and pathological features of AS are very similar, so the apoE-/- mouse is study on the pathogenesis of AS. Reveal excellent animal model of AS occurrence and development mechanism.
However, in apoE-/- mice AS occurred early, mild lesions, less interference under the condition that the sequence detecting inflammation related genes expression system is the interaction between the difference and the genes have seldom been reported. Therefore, this experiment using the mouse model of early detection of AS mice aorta, sequential expression of various inflammatory related genes in the liver and spleen, to reveal the risk factors in early stage plays a key role in the occurrence of AS, and to explore its mechanism of action, for the further development of the molecular biology research foundation.
In this experiment, apoE-/- mice with the same genetic background (C57BL/6J) of wild type (wild type WT) mice as experimental subjects, using semi quantitative RT-PCR and Real-time RT-PCR technology, detection of apoE-/- mice in 1,2 and in March at the age of 3 and 14 days of age aortic, expression of inflammation related genes and 1,2 sequence in March at the age of 4 age in the liver and spleen; and by detection of serum biochemical indexes combined with the analysis of the aortic root and liver pathological features, explore the relationship between AS and early lesions temporal expression of inflammation related genes.
The results are as follows:
1. by semi quantitative RT-PCR in detection of aortic 11 inflammation related genes in apoE-/- compared with WT mice, 1,2 and in March at the age of IL-1 beta mRNA expression levels were significantly up-regulated.1 months of age VCAM-1, I kappa B- alpha, and the expression level of WT mice were significantly up-regulated the expression of TNF- alpha and MMP2 mRNA TGF- beta and SOD1,2 month old PDGF- alpha and CD36,3 months of age, other age groups had no significant change; and Albumin in 1 and February were significantly decreased in the liver. Detection of 7 inflammation related genes, the expression level of CRP and JAK1 mRNA in February at the age of 14 days to no significant change, in March at the age of significant upregulation of.NF- gene kappa B in each age apoE-/- mice in the aorta and liver expression and age-matched WT mice showed no significant difference. The other 3 genes detected in the liver did not change significantly compared with age-matched WT mice during 14 to March. At the age of 8 detected in the spleen In apoE-/- and WT mice, the mRNA expression levels of GM-CSF and SOD1 were significantly higher than those in WT mice, but no significant changes were found in the others.
2. the use of Real-time RT-PCR in aorta was 8 inflammation related genes in March at the age of IL-1 beta expression level of mRNA and WT mice were 27 times higher than TNF-, the expression level of mRNA alpha and age-matched WT mice compared to an increase of around 3 times and 2 times respectively in January and March at the age of age, in March at the age of MCP-1 gene an increase in the expression level of about 3 times in January March at the age of age and the expression level of ICAM-1 gene was significantly increased by about 3 times, in January March at the age of age and the expression level of VCAM-1 gene was significantly increased by about 23 times and 2 times in January March at the age of age and the expression level of GM-CSF gene in WT mice compared with their peers were raised, NF- K. B and I kappa B- alpha gene had no significant change in the age of 1 and March.
The levels of TC, LDL-C and HDL-C in serum of apoE-/- mice from 3.14 days to 3 month old, 4 age groups were significantly higher than those of the same age WT mice. TG level increased significantly from 1 month old to the same age WT mice, and showed a significant upward trend with the age of mice.
4.1 month old apoE-/- mice aortic intimal lipid deposition appeared slightly visible under the light microscope, the deposition area is small, mainly distributed in the vicinity of the aortic valve, with the increase of age thediseasegraduallyincrease, in March at the age of lipid deposition area becomes larger, the deposition quantity is increased, not only limited in the valve.
5. morphological changes of liver were observed in apoE-/- mice from the beginning of January age, liver cells appeared under light microscope, the fat droplets, fat droplets are smaller and less in quantity, with the age of mice increase fat droplets gradually become larger, the number of changes, until the formation of vacuoles, showed significant accumulation of lipid in liver cells and fatty liver disease.
The experimental results show that, with aging, AS lesions gradually increased, compared with WT mice inflammation related genes, expression of the timing difference, the temporal expression of inflammation related genes differentially is downstream gene NF- B signaling pathway, constitute a complex regulatory network with NF- kappa B as the core, interaction and the mutual influence between them, participate in the chronic inflammatory process, play an important role in the occurrence and development of apoE-/- in the early mouse AS.

【学位授予单位】：山东师范大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R-332

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