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研究生:陳軍
研究生(外文):Chun Chen
論文名稱:探討第二型髓細胞觸發受體在動脈粥狀硬化中調控泡沫細胞生成的機制
論文名稱(外文):Investigation of the Triggering Receptor Expressed on Myeloid cells 2-mediated mechanism involved in foam cell formation in atherosclerosis
指導教授:陳念榮
指導教授(外文):Nien-Jung Chen
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:微生物及免疫學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2017
畢業學年度:105
語文別:英文
論文頁數:51
中文關鍵詞:巨噬細胞過氧化物酶體增殖物活化受體第二型觸發反應受體CD36動脈粥狀硬化氧化態低密度脂蛋白泡沫細胞
外文關鍵詞:macrophagePPAR-γTREM-2CD36Atherosclerosisox-LDLfoam cells
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動脈粥狀硬化(Atherosclerosis)是一種慢性發炎的疾病,且被認為參與在多種不同的疾病中。過往的研究已經指出泡沫細胞(Foam cell)在動脈粥狀硬化的形成過程中扮演一個重要的角色,且泡沫細胞的形成過程與氧化態低密度脂蛋白(oxidized low-density lipoprotein)有很大的關連性。我們實驗室在過往已經發現了第二型骨髓細胞觸發受體(TREM-2)會參與泡沫細胞的形成,失去了第二型骨髓細胞觸發受體會降低泡沫細胞的形成。另一方面,過往研究已經發現了脂肪酸轉位酶 (fatty acid translocase, FAT/CD36)對於泡沫細胞的生成也是扮演非常重要的角色。特別的是,我們實驗室先前已經發現了TREM-2會影響CD36的mRNA的表現量。我們首先探討不同基因型巨噬細胞的CD36是否會有差異。實驗發現TREM-2基因缺失的巨噬細胞有較低量的CD36,因此接著分析γ型氧化物酶體增殖物活化受體 (PPAR-γ),而結果也是TREM-2基因缺失的巨噬細胞有較低量的PPAR-γ,但是基因表現上不論是野生型(Wild Type)或是TREM-2基因缺失的巨噬細胞是沒有差異的。再者,我們也發現了使用兩種不同的刺激物來刺激TREM-2後,野生型會比TREM-2基因缺失的巨噬細胞有較高的PPAR-γ表現量。我們同時也有使用中和CD36的抗體來阻擋CD36與氧化態低密度脂蛋白結合,而結果顯示TREM-2有參與在誘發CD36產生的PPAR-γ訊號傳遞中。根據過往的實驗結果顯示磷酸化的胞外訊息調控激酶(phosphorylation ERK)會調控PPAR-γ的活性,而其機制是因為phosphorylated ERK會磷酸化PPAR-γ使其活性降低,因此我們也探討並確認了TREM-2基因缺失的巨噬細胞比野生型有較高的phosphorylation ERK。最後我們將會以U0126來確認TREM-2是藉由ERK的磷酸化影響PPAR-γ的表現。統整以上的結果,我們發現了TREM-2會影響CD36的表現量,而其機轉是因為TREM-2基因缺失的巨噬細胞有較低的PPAR-γ表現量,再更進一步的探討,我們發現了低量的PPAR-γ很有可能是因為TREM-2基因缺失的巨噬細胞有較高的phosphorylated ERK。
Atherosclerosis is a chronic inflammatory disorder and have been showed involved in many disease. Recent studies have shown that foam cells play an important role in the development of atherosclerosis. The foam cells are associated with macrophages and the oxidized low-density lipoprotein (ox-LDL). Our lab had shown that triggering receptor expressed on myeloid cells type II (TREM-2) was a crucial receptor for foam cell formation. These unpublished data suggested that the formation of foam cells was reduced in ox-LDL treated macrophages lacking TREM-2. Otherwise, CD36 have been also reported to involve in foam cells formation when the macrophage uptake ox-LDL. Interestingly, our preliminary result suggested that losing TREM-2 induced a lower level gene expression of CD36, but not SA-A. We first investigated the surface CD36 level. Our data implicated that TREM-2 deficiency led to a low level of CD36 expression in macrophage. We then analyzed the PPAR-γ and found that TREM-2 depleted BMDM express a lower level of PPAR-γ protein, but not mRNA level. We further confirmed that a higher PPAR-γ protein expression in the WT BMDM compared to TREM-2 depleted BMDM after treat with the serum or ox-LDL. We also used anti-CD36 antibody to block the binding between CD36 and ox-LDL. These data suggested that TREM-2 involved in the CD36 mediated PPAR-γ signals pathway. Since some papers suggested that phosphorylated ERK involved in the PPAR-γ activity because the PPAR-γ may act as a substrate for ERK activity, we at last confirmed that a lower level of phosphorylation ERK in the WT BMDM compared to TREM-2 depleted BMDM after treat with the serum or ox-LDL. We will use the U0126 to dissect the recover assay. In conclusion, we showed that losing TREM-2 may led CD36 downregulation, and these may due to a lower PPAR-γ expression since TREM-2 depleted BMDM have a higher phosphorylation ERK.
誌謝 I
摘要 II
Abstract IV
Table of Content VI
Abbreviations IX
Introduction 1
1. Atherosclerosis 1
2. Foam cells formation and 2
3. TREM family 3
4. TREM-2 4
5. Scavenger receptor 5
6. The regulation of CD36 via PPAR-γ 5
Hypothesis and Specific Aim 7
Material 8
1. Mouse Strain 8
2. Cell Line 8
3. Cells Culture Medium 8
4. Bone marrow isolation and differentiation: 9
5. BV-2 cell line culture and harvest 9
6. Oxidized Low-Density Lipoprotein preparation 10
7. Fluorescence staining for Dil-Oxidized Low-Density Lipoprotein 10
8. Oil red O (ORO) staining: 10
9. Flow Cytometry reagents 10
10. mRNA Reverse Transcription reagents and qPCR reagents 11
11. Primer for Real-Time PCR mRNA Reverse Transcription reagents 11
12. Western Blot reagents 12
13. Other reagents 14
14. Antibody 14
Method 16
1. Bone marrow derived macrophage (BMDM) isolation and culture 16
2. BV2 cell line and culture 16
3. Ox-LDL preparation 17
4. Foam cells formation 17
5. Old red O staining 17
6. Fluorescence staining 18
7. FACS assay 19
8. RNA isolation and qPCR assay 19
9. Western Blotting 20
10. Statistical analysis 20
Results 22
1. Absence of TREM-2 in macrophage leads to a reduced level of ox-LDL uptake which associate with a reduction of the CD36 expression 22
2. TREM-2 deficiency associates with a decrease in PPAR-γ expression 23
3. TREM-2 is crucial in ox-LDL- and Serum-mediated PPAR-γ activation 24
4. TREM-2 involved in the CD36 mediated PPAR-γ signals pathway 25
5. TREM-2 regulates ERK phosphorylation pathway to downregulate the PPAR-γ expression 25
6. Inhibit the phosphorylation of ERK did not restore the PPAR-γ level in TREM-2 -/- BMDM 26
Discussion 27
References 31
Figures 39
Figure 1. TREM-2 deficiency leads to a low level of CD36 expression in macrophage. 40
Figure 2. TREM-2 KD express lower MHC II than the WT BMDM. 42
Figure 3. TREM-2 KD leads to a low level of CD36 expression in microglia cell line. 43
Figure 4. Depletion of TREM-2 downregulate the PPAR-γ in translation level. 44
Figure 5. TREM-2-/- BMDM showed a lower PPAR-γ after treat the medium with FBS. 45
Figure 6. Anti-CD36 antibody decrease the PPAR-γ expression after treat the medium with ox-LDL. 48
Figure 7. A higher phosphorylation form ERK in TREM-2-/- than WT BMDM in response to the medium with FBS 49
Figure 8. The PPAR-γ did not restored after inhibit the phosphorylation of ERK. 50
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