臺灣博碩士論文加值系統

嗜酸性白血球的趨化能力及顆粒蛋白在過敏性呼吸道疾病具有重要的特色。我們建立了利用HL-60 clone 15 (HC15)細胞株衍生的替代細胞模型，藉由在鹼性環境下使用丁酸刺激HC15使之衍生似嗜酸性白血球特性。降血脂藥物辛伐他酊可透過甲羥戊酸途徑抑制在嗜酸性白血球中由介白質-5誘發的C-C趨化因子受體3(CCR3)表現量及相關的趨化能力，在人類週邊血液嗜酸性白血球也可觀察到相同現象。透過我們建立的過敏動物模式，我們發現給予大鼠口服辛伐他酊可抑制其鼻腔黏膜中嗜酸性白血球的浸潤。在細胞及動物模式中，我們發現辛伐他酊能降低嗜酸性白血球細胞數及ECP總表現量；經細胞數效正後，辛伐他酊並未明顯抑制ECP的製造與釋放。進一步分析發現辛伐他酊會透過誘發細胞週期停滯在G1/S期而抑制了HC15細胞的增生，此抑制能力與甲羥戊酸途徑有關的細胞週期相關週期素A表現減少相關。於是我們得知辛伐他酊可使ECP總表現量減少，並非抑制其製造或分泌，而是經甲羥戊酸途徑抑制了嗜酸性白血球的增生所造成。若經由適當的給予途徑及劑量，辛伐他酊也許能提供經常規治療仍無效的過敏性呼吸道疾病另一項治療選擇。

Chemotaxis of eosinophils and their granular proteins are important features of allergic airway inflammatory diseases. We established the HL-60 clone 15 (HC15) cells as a feasible alternative cell model for human primary eosinophils. Eosinophils could be derived by treating HC15 cells with butyric acid (BA) in an alkaline condition or through direct isolation from human peripheral blood. Simvastatin, a cholesterol-lowering agent, was demonstrated to inhibit IL-5-induced CC chemokine receptor 3 (CCR3) expression and chemotaxis of eosinophils mediated via the mevalonate pathway. Similar results were also exhibited in human primary eosinophils. In vivo animal models further confirmed that oral simvastatin could significantly suppress the infiltration of eosinophils into turbinate tissues. Both HC15 cell counts and eosinophil cationic protein (ECP) levels decreased after simvastatin treatment in the animal and cell models; however, after a cell count adjustment, simvastatin was not observed to exert a significantly inhibitory effect on ECP intracellular formation or release. Instead, cell cycle analysis showed that simvastatin inhibited the proliferation of HC15 cells through the mevalonate pathway by inducing G1/S cell cycle arrest in a dose-dependent manner with cyclin A involvement. Collectively, statins may serve to have anti-eosinophilic inflammation effects if applied to the adequate route and at the correct dosage and may provide another treatment option for those whose allergic airway diseases are refractory to standard therapies.

Table of Contents
Recommendation Letter from the Thesis Advisor………………………………..……..…….
Thesis/Dissertation Oral Defense Committee Certification………………………….………..
Acknowledgments………………………………………………………………………….…. iii
Chinese Abstract……………………………………………………………………...……….. iv
English Abstract………………………………………………….…………………………..... v
Chapter 1 Introduction …………………………………………………………………………. 1
1.1 The impact of atopic diseases: Allergic rhinitis and asthma…………………………... 1
1.2 The link between upper and lower airway atopic diseases……………………………. 4
1.3 Allergic reaction and the role of eosinophils……………………………………….…... 6
1.4 The importance of eosinophilic granule proteins………………………………………. 9
1.5 The impact of eosinophil cationic protein (ECP)………………………………………. 10
1.6 Cytokines and chemokines involved in eosinophilic response………………………… 13
1.7 To seek the alternative cell culture……………………………………………………... 17
1.8 The possible roles of statins in airway disease…………………………………………. 17
1.9 The study objectives………………………………………………………………..…. 20
Chapter 2 Materials and Methods: To Establish The Cell Model and The Animal Model…….. 22
2.1 Reagents…………………………………………………………………..……………. 22
2.2 Prepare the cell cultures……………………………………………………..…………. 23
2.3 Verify the best differentiation condition for the cell culture ………………………...… 24
2.4 RNA extraction and RT/real-time polymerase chain reaction……………….................. 25
2.5 Cell culture condition determination…………………………...................................…. 29
2.6 Staining of cellular granular proteins………………………………………………...… 31
2.7 Western blotting……………………………………………………………………..…. 32
2.8 Immunocytochemical stain ……………………………………………….………….... 33
2.9 Preparation of human primary eosinophils…………………………………………..… 33
2.10 Chemotaxis assay…………………………………………………….….………….… 35
2.11 Animals preparation……………………………………………………………..….… 36
2.12 Protocol of allergen sensitization and challenge…………………………………....… 37
2.13 Animal blood sample collection and nasal tissue preparation……….………….……. 39
2.14 Enzyme-linked immunosorbent assay ……………………………………………...… 40
2.15 Cell cycle analysis………………………………………………………………...…... 41
2.16 Microarray analysis…………………………………………………………………… 41
2.17 Statistical analysis…………………………………………………………………..… 42
Chapter 3 Simvastatin inhibits IL-5 induced and CCR3-associated chemotaxis of eosinophils... 43
3.1 Relationship between IL-5 and CCR3 on HC15 cells………………………………….. 43
3.2 Time course of IL-5 receptor and CCR3 on HC15 cells…………………………...…... 45
3.3 Migration and chemotaxis assay of HC15 …………………………………………..… 46
3.4 IL-5 enhanced chemotaxis and CCR3 expression in HC15 cells…………………….… 48
3.5 Simvastatin effect on IL-5 induced chemotaxis of HC15 cells…………………….…... 49
3.6 Simvastatin effect on IL5R and CCR3……………………………………………….… 50
3.7 Simvastatin inhibited CCR3 through ERK pathway…………………………….……... 51
3.8 Replacement of mevalonate…………………………………………………………..... 52
3.9 Application the simvastatin effect on human primary eosinophils…………………..… 54
3.10 Histological examination for allergic rats……………………...................................... 55
3.11 Protocol of simvastatin treatment before allergen challenge………………………..... 57
3.12 The microscopic change of rat nasal tissue………………………………………..….. 59
3.13 Simvastatin effect on infiltration of eosinophils in nasal tissue………………….…… 61
3.14 Microarray analysis …………………………………………………………….…..… 62
3.15 Pathway analysis……………………………………………………………….…..…. 65
3.16 Recheck the presentation of the target genes in the cell culture…………………….... 67
Chapter 4 Simvastatin Effects on Granular Proteins and Proliferation of Eosinophils………….. 70
4.1 The presentation of granular proteins in HC15 cells after simvastatin treatment…........ 70
4.2 The presentation of ECP in rat nasal tissue………………...………………………...… 71
4.3 Simvastatin effect on ECP presentation ……………………………………………...... 74
4.4 Simvastatin effect on MBP and other eosinophilic granular protein…………………... 76
4.5 Simvastatin effect on cell cycles of other cell types………………………………….... 77
4.6 Simvastatin effect on cell cycles of HC15 cells…………………………….………….. 79
4.7 Simvastatin effect on cell cycle-associated cyclins and cdks in HC15 cells…………… 81
4.8 Mevalonate reversed the effect of simvastatin on the cell cycle and the associated cyclin expression ………………………………………………………………………..............… 82
4.9 Simvastatin effect on cell apoptosis analyzed by flow cytometry……………………….. 84
4.10 Possible signaling pathway involved for the effect of simvastatin………………….... 85
4.11 Simvastatin as a feasible therapeutic agent for allergic airway diseases….…………. 85
Chapter 5 Conclusion and Future Study……………………………………………………...….. 87
5.1 Application of the cell culture and animal model…………………………………....… 87
5.2 Effect of lactoferrin on allergic diseases……………………...…………………….….. 87
5.2.1 Effect of lactoferrin on HC15 cells …………………………………………..….. 88
5.2.2 Effect of lactoferrin on chemotaxis of HC15 cells…………………………….…. 89
5.2.3 Effect of lactoferrin on granular proteins of HC15 cells……………………..…... 90
5.2.4 Target genes obtained from microarray in lactoferrin study………………….….. 93
5.2.5 Future goals for verifying therapeutic ability of LF for allergic diseases………... 95
5.3 Conclusions and future plans………………………………..……………………….… 97

References………………………………………………………………………………...……. 101

Lists of Figures
Figure 1-1 The global prevalence of allergic rhinitis.……………………………...……...…….. 1
Figure 1-2 Economic costs in allergic rhinitis……………………………………………..….... 2
Figure 1-3 Allergic rhinitis and asthma have similar prevalence patterns………..….................… 3
Figure 1-4 Allergic rhinitis (AR) and its possible co-morbidities……………………………..… 3
Figure 1-5 Correlation between predicted forced expiratory volume in one second (FEV1) value (%) and severity of sinusitis in asthmatics ………………..…………………………………………... 4
Figure 1-6 Pathophysiological association for the concept of united airway disease…………….. 5
Figure 1-7 The immunohistochemistry stain of nasal mucosa in asthmatic patients……............... 6
Figure 1-8 Role of eosinophils in an allergic reaction……………………………………………. 8
Figure 1-9 The biology and function of major eosinophilic granular proteins…………..……….. 9
Figure 1-10 The interaction between endothelial cells and eosinophils during the chemotaxis process …………………………………………………………………………………………... 14
Figure 1-11 Cytokines and chemokines that related to the characteristics of eosinophils during the chemotaxis process and allergic reaction…………………………………………………..….… 15
Figure 1-12 Clone 15 HL-60 cells in microscopic view (200×)……………………..…….….… 17
Figure 1-13 Schematic diagram of the mevalonate pathway for cholesterol synthesis…...…..… 18
Figure 1-14 Eosinophilic inflammation in airway could be inhibited by simvastatin treatment in an allergic murine model………………………………………………………………………...….. 19
Figure 2-1 Naïve Clone 15 HL-60 (HC15) cells before histone deacetylase inhibitors treatment... 23
Figure 2-2 The comparison of cell viability of HC15 cells in time course with / without condition medium changed every 2 days.……………………………………….……………………..…... 25
Figure 2-3 The time course of mRNA presentations of specific markers for HC15 cells treated with BA analyzed by PCR………………………………………………………………….……….… 28
Figure 2-4 The mRNA presentation of specific markers for HC15 cells treated with BA and NaBu analyzed by PCR………………………………………………………………………....…….... 28
Figure 2-5 Clone 15 HL-60 cells in different stains in microscopic view………………………. 30
Figure 2-6 The flow chart of the set condition for the cell culture………………….………….... 31
Figure 2-7 Fast green and neutral red stain before and after BA treatment of HC 15 cells…..…. 32
Figure 2-8 The human eosinophil enrichment kit- EasySepTM………………………………..… 34
Figure 2-9 Chemotaxis and migration assay for HC15 cells……………………………...…..… 36
Figure 2-10 The set of OVA challenge in the allergic rat model………………………………... 38
Figure 2-11 The experimental protocol of the allergic rat model……………………..………… 39
Figure 3-1 Presentation of eosinophilic markers and receptors after IL-5 stimulation in NaBu- or BA-treated HC15 cells.…………………………………………………………...……………... 44
Figure 3-2 Presentation of eosinophilic markers in BA-treated HC15 cells after different IL-5 stimulation timing…………………………………………………………………………...….... 44
Figure 3-3 Effects of butyric acid on HC15 cells………………………………………...…....… 46
Figure 3-4 The microscopic view of the Transwell chamber filter under after Liu stain…........... 47
Figure 3-5 The results of chemotaxis and migration assay after IL-5 stimulation for HC15 cells……………………………………………………………………………………………... 47
Figure 3-6 Dose-dependent effect of IL-5 on the chemotaxis and CCR3 expression in HC15 cells..……………………………………………………………………………………………. 48
Figure 3-7 The effects of simvastatin on survival and chemotaxis of HC15 cells………….…… 49
Figure 3-8 The effects of simvastatin on IL-5 receptor expressions of HC15 cells……….......… 50
Figure 3-9 The effects of simvastatin on CCR3 expressions of HC15 cells………….……....…. 51
Figure 3-10 Effect of simvastatin treatment on downstream activation of ERK1/2 and p38 in HC15 cells……………………………………………………………………………………..………... 52
Figure 3-11 Mevalonate replacement reversed the inhibitory effects of simvastatin on HC15 cells………………………………………………………………………………………………. 53
Figure 3-12 Effects of simvastatin on the chemotaxis and CCR3 expression in human primary eosinophils……………………………………………………………………………………….. 54
Figure 3-13 The intra-nasal structural anatomy of the SD rat…………..…...………………..…. 56
Figure 3-14 OVA challenge increased eosinophil infiltration in a rat model of allergic rhinitis.... 57
Figure 3-15 The experimental protocol of simvastatin treatment before 3-day OVA challenge in the allergic rat model………………………………………………………………………..……….. 58
Figure 3-16 The histological examination of nasal turbinate tissue in the allergic rat model….... 60
Figure 3-17 Simvastatin reduced eosinophil infiltration in a rat model of allergic rhinitis……... 61
Figure 3-18 Microarray analysis results for HC15 cells before and after simvastatin treatment (1).63
Figure 3-19 Microarray analysis results for HC15 cells before and after simvastatin treatment (2).64
Figure 3-20 Representation of Gene Ontology analysis of the target genes.…….…………..… 66
Figure 3-21 The pathway analysis of the target genes…………………………………………. 66
Figure 3-22 The expression of (A) NGEF, (B) MAP3K15 and (C) FRMD mRNA levels analyzed by real-time PCR……………………………………………………………………………………. 68
Figure 4-1 Simvastatin effect on the formation of eosinophilic granular proteins…………........ 70
Figure 4-2 Eosinophil cationic protein (ECP) expression in nasal tissue in the allergic animal model.………………………………………………………………………………..………...… 73
Figure 4-3 Eosinophil cationic protein (ECP) expression did not differ after simvastatin treatment..………………………………………………………………………………………... 75
Figure 4-4 Major basic protein (MBP) expression in nasal tissue in the allergic animal model… 76
Figure 4-5 Expressions of other eosinophil granular proteins did not differ after simvastatin treatment…………………………………………………………………………………………. 77
Figure 4-6 The cell cycle was altered after simvastatin treatment.………………………….…... 80
Figure 4-7 Simvastatin inhibited the expression of cyclin A………………………………...….. 81
Figure 4-8 Mevalonate supplementation reversed the effect of simvastatin……………..…….... 83
Figure 4-9 Mevalonate replacement reversed simvastatin- induced cell apoptosis…………...… 84
Figure 5-1 The effects of lactoferrin (LF) on CCR3 expressions of HC15 cells……………...… 88
Figure 5-2 The effects of lactoferrin (LF) on chemotaxis of HC15 cells…………..……….…… 90
Figure 5-3 The effects of lactoferrin (LF) on the presentation of ECP in HC15 cells…….…..… 92
Figure 5-4 The effects of lactoferrin (LF) on the presentation of ECP in HC15 cells…...……… 92
Figure 5-5 Results of microarray analysis for LF pre-treatment protocol …..…………….…..… 94
Figure 5-6 The expression of NGEF, MAP3K15 and FRMD1 mRNA levels without and with lactoferrin (LF) pre-treatment in HC15 cells analyzed by real-time PCR…………………….… 95
Figure 5-7 The protocol of the allergic rat model for lactoferrin (LF) ………………………..… 96
Figure 5-8 Schematic diagram of our proposed mechanism for simvastatin effects on eosinophil behaviors…………………………………………………………………………………..…...… 99

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