本文關(guān)鍵詞：ApoE缺失小鼠炎性因子關(guān)鍵基因的表達(dá)研究　出處：《山東師范大學(xué)》2008年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 載脂蛋白E基因缺失小鼠 炎癥相關(guān)基因 動(dòng)脈 肝臟 脾臟

【摘要】： 心腦血管疾病正嚴(yán)重威脅著人類的健康,由其導(dǎo)致的死亡率呈逐年上升趨勢(shì),動(dòng)脈粥樣硬化(atherosclerosis, AS)是其病理基礎(chǔ)。關(guān)于AS的發(fā)病機(jī)制存在多種學(xué)說(shuō),主要是由多種因素引起的慢性炎癥反應(yīng)學(xué)說(shuō)以及脂代謝紊亂學(xué)說(shuō),而在機(jī)體內(nèi)慢性炎癥反應(yīng)和脂代謝紊亂相互影響、共同參與并促進(jìn)了AS發(fā)生發(fā)展。 載脂蛋白E基因缺失(apoE-/-)小鼠是目前常用的研究AS的模型,在普通飲食條件下可自發(fā)產(chǎn)生AS,隨著小鼠年齡的增長(zhǎng)病變逐漸加重,并且其AS病變過(guò)程中呈現(xiàn)出的病理特征與人AS非常相似,因此apoE-/-小鼠是研究AS發(fā)生機(jī)制,揭示AS發(fā)生發(fā)展機(jī)理的優(yōu)良動(dòng)物模型。 然而在apoE-/-小鼠AS發(fā)生早期,病變較輕、干擾因素較少的情況下,較系統(tǒng)的檢測(cè)炎癥相關(guān)基因的時(shí)序表達(dá)差異以及各基因之間的相互作用的報(bào)道尚不多見(jiàn)。為此,本實(shí)驗(yàn)利用該小鼠為模型,檢測(cè)AS早期小鼠主動(dòng)脈、肝臟和脾臟中多種炎癥相關(guān)基因的時(shí)序表達(dá),旨在揭示在AS發(fā)生早期起關(guān)鍵作用的危險(xiǎn)因子,并初步探討其作用機(jī)理,為進(jìn)一步深入開展分子生物學(xué)研究奠定基礎(chǔ)。 本實(shí)驗(yàn)選用apoE-/-小鼠和具有相同遺傳背景(C57BL/6J)的野生型(wild type, WT)小鼠為實(shí)驗(yàn)對(duì)象,利用半定量RT-PCR以及Real-time RT-PCR技術(shù),檢測(cè)apoE-/-小鼠在1、2和3月齡3個(gè)年齡段主動(dòng)脈以及14天、1、2和3月齡4個(gè)年齡段肝臟和脾臟中炎癥相關(guān)基因的時(shí)序表達(dá)特點(diǎn);并通過(guò)血清生化指標(biāo)的檢測(cè)結(jié)合主動(dòng)脈根部和肝臟病理形態(tài)學(xué)特征的分析,探討炎癥相關(guān)基因的時(shí)序表達(dá)與AS早期病變的關(guān)系。 通過(guò)實(shí)驗(yàn)得到如下結(jié)果: 1.利用半定量RT-PCR在主動(dòng)脈檢測(cè)的11條炎癥相關(guān)基因中,apoE-/-與WT小鼠相比,1、2和3月齡時(shí)IL-1β的mRNA表達(dá)水平均顯著上調(diào)。1月齡時(shí)VCAM-1、IкB-α、TGF-β和SOD1,2月齡時(shí)PDGF-α和CD36,3月齡時(shí)TNF-α和MMP2的mRNA表達(dá)水平與同齡WT小鼠相比均顯著上調(diào),其它年齡段無(wú)顯著變化;而Albumin在1和2月齡時(shí)顯著降低。在肝臟檢測(cè)的7條炎癥相關(guān)基因中,CRP和JAK1的mRNA表達(dá)水平在14天到2月齡時(shí)無(wú)顯著變化,3月齡時(shí)顯著上調(diào)。NF-кB在各年齡段apoE-/-小鼠主動(dòng)脈和肝臟中的基因表達(dá)量與同齡WT小鼠相比均無(wú)顯著差異。在肝臟中檢測(cè)的其它3條基因在14天至3月齡期間與同齡的WT小鼠相比并無(wú)顯著變化。在脾臟中檢測(cè)的8條炎癥相關(guān)基因中,apoE-/-與WT小鼠相比,2月齡時(shí)GM-CSF和SOD1的mRNA表達(dá)水平顯著升高,其它均無(wú)顯著變化。 2.利用Real-time RT-PCR在主動(dòng)脈檢測(cè)的8條炎癥相關(guān)基因中,3月齡時(shí)IL-1βmRNA表達(dá)水平與同齡WT小鼠相比升高約27倍,TNF-αmRNA在1月齡和3月齡時(shí)表達(dá)水平與同齡WT小鼠相比分別升高約3倍和2倍,3月齡時(shí)MCP-1基因表達(dá)水平升高約3倍,1月齡和3月齡時(shí)ICAM-1基因表達(dá)水平均顯著上調(diào)約3倍,1月齡和3月齡時(shí)VCAM-1基因表達(dá)水平均顯著上調(diào)約23倍和2倍,1月齡和3月齡時(shí)GM-CSF基因表達(dá)水平與同齡WT小鼠相比均上調(diào),NF-кB和IкB-α基因在1和3月齡時(shí)均無(wú)顯著變化。 3. 14天至3月齡4個(gè)年齡段的apoE-/-小鼠血清TC、LDL-C和HDL-C水平均顯著高于同齡WT小鼠;TG水平從1月齡開始比同齡WT小鼠顯著升高,并隨小鼠年齡的增長(zhǎng)呈現(xiàn)明顯的上升趨勢(shì)。 4. 1月齡apoE-/-小鼠主動(dòng)脈內(nèi)膜出現(xiàn)光鏡下可見(jiàn)的輕微的脂質(zhì)沉積,沉積面積較小,主要分布在主動(dòng)脈瓣膜附近,隨年齡增長(zhǎng)病變逐漸加重,到3月齡時(shí)脂質(zhì)沉積面積變大、沉積的數(shù)量變多,也不僅僅局限在瓣膜處。 5.肝臟病理形態(tài)切片觀察發(fā)現(xiàn)apoE-/-小鼠從1月齡開始,肝臟細(xì)胞內(nèi)出現(xiàn)光鏡下可見(jiàn)的脂肪滴,脂肪滴較小且數(shù)量較少,隨小鼠年齡增長(zhǎng)脂肪滴逐漸變大,數(shù)量變多,直至形成空泡,表現(xiàn)出明顯的肝細(xì)胞脂質(zhì)堆積和肝臟脂肪性病變。 以上實(shí)驗(yàn)結(jié)果顯示,隨著小鼠年齡的增長(zhǎng),AS病變逐漸加重,炎癥相關(guān)基因與WT小鼠相比,出現(xiàn)時(shí)序性差異表達(dá),這些時(shí)序表達(dá)差異的炎癥相關(guān)基因是NF-κB信號(hào)通路的上下游基因,構(gòu)成了以NF-κB為核心的復(fù)雜調(diào)控網(wǎng)絡(luò),它們之間相互作用、相互影響,共同參與慢性炎癥過(guò)程,在apoE-/-小鼠AS的早期發(fā)生發(fā)展中發(fā)揮重要作用。
[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.

【學(xué)位授予單位】：山東師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R-332

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本文編號(hào)：1419335