跳到主要內容

臺灣博碩士論文加值系統

(44.212.96.86) 您好!臺灣時間:2023/12/06 15:54
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

: 
twitterline
研究生:賴梅玉
研究生(外文):Mei-Yu Lai
論文名稱:SP-D基因變異性與兒童氣喘關係
論文名稱(外文):Association of the genetic polymorphism of the surfactant protein (SP)-D with childhood asthma
指導教授:王志堯謝奇璋謝奇璋引用關係
指導教授(外文):Jiu-Yao WangChi-Chang Shieh
學位類別:碩士
校院名稱:國立成功大學
系所名稱:分子醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:英文
論文頁數:66
中文關鍵詞:基因變異性肺泡表面蛋白D氣喘
外文關鍵詞:asthmaSNPSP-D
相關次數:
  • 被引用被引用:1
  • 點閱點閱:206
  • 評分評分:
  • 下載下載:10
  • 收藏至我的研究室書目清單書目收藏:1
肺臟是我們體內氣體交換的器官,所以肺臟經常要接觸空氣中的病原菌、過敏原和污染物質,肺臟
內的防禦系統必須要有效且快速的清除這些病原菌。肺泡的先天免疫防禦系統機制包含有噬中性白血球、
巨噬細胞和肺泡表面組成物質。其中一個肺泡表面物質是肺泡表面蛋白D(SP-D),SP-D在肺泡表面可藉
著鈣離子的幫助來清除大多數的病原菌,同時還可抑制過敏原及氣管的發炎反應。目前已知在SP-D的
coding sequence中有三個單一核甘酸變異點(SNP),分別是位在N端的SP-D/11(Thr11Met),
collagen-like domain的SP-D/160(Thr160Ala),及CRD的SP-D/270(Ser270Thr)。在先前的研究中發現,
SP-D/11的基因型和某些疾病有相關性,如在墨西哥族群中,帶有SP-D/11CC (Thr11Thr)基因型的人比較
容易感染結核桿菌。然而,帶有SP-D/11TT (Met11Met)基因型的嬰兒反而會比較容易受到呼吸道融合病
毒的感染。除此之外,帶有SP-D/11CC (Thr11Thr)基因型的人其血清中的SP-D濃度會比帶有SP-D/11TT
(Met11Met)基因型的人還要來的低。同時,也因為SP-D在肺泡的先天免疫防禦系統中扮演的角色,我們
認為SP-D的基因型應該和氣喘發生的有無有一定的相關性。故在本篇研究中,我們想要知道SP-D基因型
的不同是否會影響氣喘及家庭塵蟎過敏(Der p)的發生率。首先,我們收集281個檢體,依據臨床診斷和
過敏原測試結果,把他們區分為4個不同的族群,分別是1.過敏性氣喘、2.過敏性但是不具有氣喘、3.非
過敏性氣喘和4.不過敏也沒有氣喘的個體。SP-D基因型是利用SSP-PCR(sequence specific primer-PCR)
來定出,並利用酵素免疫分析法(ELISA)來分析血清中SP-D的濃度。我們發現帶有Thr11Thr基因型的人血
清中SP-D的濃度明顯低於帶有Met11Met基因型的人。若比較氣喘組與對照組或是對家庭塵蟎過敏組與
對照組基因型分佈是否有差異,則發現只有SP-D/160這個基因型在對家庭塵蟎過敏組及對照組的分佈有
差異。另外,利用單元型分析(haplotype analysis),我們發現C11/A160 haplotype在塵蟎過敏中扮演
保護的角色同時其血清中的SP-D濃度也明顯高於T11/G160 haplotype的個體。同時,帶有C11/A160
haplotype的個體,其血清中SP-D和塵蟎過敏原結合的能力明顯低於帶有T11/G160 haplotype的個體。
為了更進一步的研究不同基因型的SP-D和塵蟎過敏原結合的能力是否有所不同,我們必須要建構及表現
不同基因型的SP-D重組蛋白。截至目前為止,我們已經藉由哺乳細胞表現SP-D T11/G160的重組蛋白。
總而言之,我們認為SP-D的基因型與家庭塵蟎過敏原是有相關性的。
As a gas-exchange organ, lung is inevitably exposed to air that is contaminated with
pathogens, allergens and pollutants. Hence, the host-defense mechanism within the lung is
vital to facilitate clearance of inhaled pathogens as well as to prevent persistent
inflammation. The innate immune components in the alveolar of lung alveolar include
neutrophils, alveolar macrophages, and surfactant components. One of the surfactant
proteins in the alveolar and bronchial epithelium is lung surfactant protein D
(SP-D), which belongs to the collectin family of calcium-dependent lectin with multiple
roles in innate immune defense and the regulatory function in allergen sensitization and
airway inflammation. There are three single nucleotide polymorphisms (SNPs) in the coding
sequence of human SP-D at positions 11 (Thr11Met) in the N-terminal region, 160 (Thr160Ala)
in the collagen-like domain, and 270 (Ser270Thr) in the carbohydrate recognition domain.
In the previous studies, the Thr11Thr genotype was suggested to increase the susceptibility
to tuberculosis in a Mexican population, whereas the Met11Met genotype was associated with
increased susceptibility of respiratory syncytial virus infection in infants. Besides,
individuals with the Thr11Thr genotype have significantly lower SP-D serum levels as
compared with Met11Met genotype carriers. Due to its physiological function in the innate
immunity, SP-D represents an ideal candidate gene for bronchial asthma. In this study, we
are interested whether the genetic variants of SP-D are in association with clinical
phenotypes of asthma and house dust mites, Dermatophagoides ptyronysinnus (Der p) allergen
sensitization. We have enrolled 281 school-age children in Taiwan, and divided into 4
subgroups (Allergic Asthma, Non-allergic Asthma, Allergic Non-asthma, and controls)
according to their clinical symptoms and host dust mite sensitization. SP-D SNPs was
genotyped by a sequence specific primer-PCR methodology and their serum SP-D levels were
analyzed by ELISA. Association analyses were performed by SPSS. We found that subjects with
Thr11Thr genotype had significantly lower SP-D serum levels than Met11Met genotype.
Thr11Met variant of SP-D was not associated with mite allergy or asthma. However, T
hr160Ala variant of SP-D was associated with Der p sensitization, but not with asthma
phenotype. According to haplotype analysis, we also found that the frequencies of C11/A160
play as a protective role in mite-sensitization individuals and serum SP-D levels of
C11/A160 haplotype are higher than those of non-C11/A160 haplotype individuals. In the
further experiments, we also found that serum SP-D from C11/A160 haplotype subjects had
lower mite allergen binding ability than that of SP-D of T11/G160 haplotype. In order to
study further on the mite allergen binding affinity between sifferent genetic variants of
SP-D, we need to construct and express different genotypes of SP-D recombinant proteins.
At the present moment, we had expressed the recombinant protein with T11/G160 of full
length SP-D from mammalian cells in Flp-In system. Through the project, we find the
functional amino acid variants in SP-D may play a major role in the genetic pre-disposition
to allergen sensitization and childhood asthma.
Contents
Chinese Abstract...............................................................................................1
English Abstract……………………………………………………………....3
Contents………………………………………………………………………6
Content of figures……………………………………………………….........9
Content of tables…………………………………………………………….10
Chapter 1: Introduction………………………………………..…………….11
1. Mechanism of lung host-defense and the function of SP-D……...11 2. Protein structure and functional domains of SP-D…………….....12
3. The immunoregulatory effect of surfactant proteins in allergic
Inflammatory of asthma…………………………………………..13
4. Gene and single nucleotide polymorphisms (SNPs) of SP-D…....14
5. The polymorphism studies of the SP-D………………….………14
6. The aim of this study……………………………………..………15
Chapter 2: Materials and method………………………………………..…..17
1. Clinical sample collection……………………………………..…17
2. DNA preparation……………………………………………….....17
3. Genotyping of the Surfactant Protein D (SP-D)……………….....19
4. Determination of SP-D level in plasma………………………..…20
5. Direct binding between Derp to SP-D…………………………....22
6. Statistical analysis……………………………………………..….23
7. Transfect SP-D T11/G160 plasmid into Flp-In 293 cell………..…..24
Chapter 3: Results…………………………………………………..……….26
1. Study population……………………………………………..….26
2. Serum SP-D levels were higher in mite-sensitive children……...26
3. No significant differences of the serum SP-D levels between the asthmatics and non-asthmatics individuals……………………...27
4. There was no significant difference in the serum SP-D levels among the four study groups…………………………..………...27
5. Significant difference of serum IgE levels in the four study groups……………………………………………………………27
6. Genotyping (SP-D/11,160, and 270) the 281 samples by sequence-specific primer (SSP)-PCR method…………………...28
7. There were association between the polymorphism sites of SP-D and serum SP-D levels…………………………………………..29
8. There were no association between the genetic polymorphisms of SP-D and serum IgE levels………………………………………29
9. No correlations between serum IgE levels and serum SP-D levels..............................................................................................30
10. There were no association between the polymorphism sites of the SP-D and asthma………………………………………………...30
11. Polymorphism of SP-D/160 was associated with the mite-sensitizaiton………………………………………………..30
12. Haplotype analysis between asthma and non-asthma and mite-sensitization and controls…………………………………..31
13. Serum SP-D levels in C11/A160 haplotype and non-C11/A160 haplotype was significantly different...………………………….31
14. Mite allergen binding affinity was significantly different between SP-D C11/A160 haplotype and SP-D T11/G160 haplotype…………32
15. Expression of SP-D T11/G160 recombinant protein from Flp-In system……………………………………………………………32
Chapter 4: Discussion……………………………………………………….33
References………………………………………………………………..…37
Figures………………………………………………………………………44
Tables………………………………………………………………………..59
Appendixes………………………………………………………………….65
Appendix 1: The immunomodulatory roles of lung surfactant proteins,
SP-A and SP-D, in allergic inflammation of asthma…………..65
Appendix 2: Sequence specific primer-PCR (SSP-PCR)…………………...66















Content of Figures
Figure 1: Boxplot of the levels of serum SP-D among the mite-sensitized and non-mite sensitized individuals…………………………………..44
Figure 2: Boxplot of the levels of serum SP-D among the asthmatics and non-asthmatics individuals……………………………………….45
Figure 3: Boxplot of serum SP-D levels in different phenotypes…………...46
Figure 4: Boxplot of serum IgE levels in different phenotypes……………..47
Figure 5: Localization of the polymorphism site of human SP-D gene…….48
Figure 6: Detected SP-D/11 polymorphism localized at N-terminal domain by SSP-PCR………………………………………………………….49
Figure 7: Detected SP-D/160 polymorphism localized at N-terminal domain by SSP-PCR………………………………………………………50
Figure 8: Detected SP-D/270 polymorphism localized at N-terminal domain by SSP-PCR………………………………………………………51
Figure 9: Boxplot of serum SP-D levels in different genotypes…………….52
Figure 10: Boxplot of the levels of total serum IgE levels in different genotypes………………………………………………………....53
Figure 11: Correlation of the total serum IgE levels with serum SP-D levels……………………………………………………………...54
Figure 12: Boxplot of serum SP-D levels in mite allergen C11/A160 haplotype and non- C11/A160 haplotype………………...……………………55
Figure 13: Boxplot of serum IgE levels in C11/A160 haplotype and non- C11/A160 haplotype………………………………………………...56
Figure 14: Mite allergen binding affinity of variant genotypes of SP-D……57
Figure 15: Expression of SP-D T11/G160 protein from Flp-In system………58
Content of Tables
Table 1: PCR primers used to detect polymorphisms of the gene for SP-D...59
Table 2: Study population…………………………………………………...60
Table 3: Association of SP-D polymorphisms sites with asthma…………...61
Table 4: Association of SP-D polymorphisms sites with mite-sensitization..62
Table 5: Asthma haplotype analysis………………………………………...63
Table 6: Mite allergen haplotype analysis…………………………………..64
1.Vercelli D. Innate immunity: sensing the environment and regulating the regulators. Curr Opin Allergy Clin Immunol 2003; 3:343-6.

2.Holt PG, Macaubas C, Stumbles PA, Sly PD. The role of allergy in the development of asthma. Nature 1999; 402:B12-7.

3.Hamm H, Fabel H, Bartsch W. The surfactant system of the adult lung: physiology and clinical perspectives. Clin Investig 1992; 70:637-57.

4.Reid KBM, Clark H, Palaniyar N. Surfactant and lung inflammation. Thorax 2005; 60:620-2.

5.Wright JR. Immunoregulatory functions of surfactant proteins. Nat Rev Immunol 2005; 5:58-68.

6.Madsen J, Kliem A, Tornoe I, Skjodt K, Koch C, Holmskov U. Localization of lung surfactant protein D on mucosal surfaces in human tissues. J Immunol 2000; 164:5866-70.

7.Akiyama J, Hoffman A, Brown C, Allen L, Edmondson J, Poulain F, et al. Tissue distribution of surfactant proteins A and D in the mouse. J Histochem Cytochem 2002; 50:993-6.

8.Stahlman MT, Gray ME, Hull WM, Whitsett JA. Immunolocalization of surfactant protein-D (SP-D) in human fetal, newborn, and adult tissues. J Histochem Cytochem 2002; 50:651-60.

9.Leth-Larsen R, Nordenbaek C, Tornoe I, Moeller V, Schlosser A, Koch C, et al. Surfactant protein D (SP-D) serum levels in patients with community-acquired pneumonia small star, filled. Clin Immunol 2003; 108:29-37.

10.Reid KB. Structure/function relationships in the collectins (mammalian lectins containing collagen-like regions). Biochem Soc Trans 1993; 21:464-8.
11.Crouch E, Persson A, Chang D, Heuser J. Molecular structure of pulmonary surfactant protein D (SP-D). J Biol Chem 1994; 269:17311-9.

12.Holmskov U, Thiel S, Jensenius JC. Collections and ficolins: humoral lectins of the innate immune defense. Annu Rev Immunol 2003; 21:547-78.

13.Lim BL, Wang JY, Holmskov U, Hoppe HJ, Reid KB. Expression of the carbohydrate recognition domain of lung surfactant protein D and demonstration of its binding to lipopolysaccharides of gram-negative bacteria. Biochem Biophys Res Commun 1994; 202:1674-80.

14.Bridges JP, Davis HW, Damodarasamy M, Kuroki Y, Howles G, Hui DY, et al. Pulmonary surfactant proteins A and D are potent endogenous inhibitors of lipid peroxidation and oxidative cellular injury. J Biol Chem 2000; 275:38848-55.

15.Kishore U, Wang JY, Hoppe HJ, Reid KB. The alpha-helical neck region of human lung surfactant protein D is essential for the binding of the carbohydrate recognition domains to lipopolysaccharides and phospholipids. Biochem J 1996; 318 ( Pt 2):505-11.

16.van de Wetering JK, van Eijk M, van Golde LM, Hartung T, van Strijp JA, Batenburg JJ. Characteristics of surfactant protein A and D binding to lipoteichoic acid and peptidoglycan, 2 major cell wall components of gram-positive bacteria. J Infect Dis 2001; 184:1143-51.

17.Palaniyar N, Nadesalingam J, Clark H, Shih MJ, Dodds AW, Reid KB. Nucleic acid is a novel ligand for innate, immune pattern recognition collectins surfactant proteins A and D and mannose-binding lectin. J Biol Chem 2004; 279:32728-36.

18.Coppolino MG, Dedhar S. Calreticulin. Int J Biochem Cell Biol 1998; 30:553-8.


19.Sim RB, Moestrup SK, Stuart GR, Lynch NJ, Lu J, Schwaeble WJ, et al. Interaction of C1q and the collectins with the potential receptors calreticulin (cC1qR/collectin receptor) and megalin. Immunobiology 1998; 199:208-24.

20.Eggleton P, Lieu TS, Zappi EG, Sastry K, Coburn J, Zaner KS, et al. Calreticulin is released from activated neutrophils and binds to C1q and mannan-binding protein. Clin Immunol Immunopathol 1994; 72:405-9.

21.Vandivier RW, Ogden CA, Fadok VA, Hoffmann PR, Brown KK, Botto M, et al. Role of surfactant proteins A, D, and C1q in the clearance of apoptotic cells in vivo and in vitro: calreticulin and CD91 as a common collectin receptor complex. J Immunol 2002; 169:3978-86.

22.Wang JY, Kishore U, Lim BL, Strong P, Reid KB. Interaction of human lung surfactant proteins A and D with mite (Dermatophagoides pteronyssinus) allergens. Clin Exp Immunol 1996; 106:367-73.

23.Wang JY, Shieh CC, You PF, Lei HY, Reid KB. Inhibitory effect of pulmonary surfactant proteins A and D on allergen-induced lymphocyte proliferation and histamine release in children with asthma. Am J Respir Crit Care Med 1998; 158:510-8.

24.Malherbe DC, Erpenbeck VJ, Abraham SN, Crouch EC, Hohlfeld JM, Wright JR. Surfactant protein D decreases pollen-induced IgE-dependent mast cell degranulation. Am J Physiol Lung Cell Mol Physiol 2005; 289:L856-66.

25.Erpenbeck VJ, Malherbe DC, Sommer S, Schmiedl A, Steinhilber W, Ghio AJ, et al. Surfactant protein D increases phagocytosis and aggregation of pollen-allergen starch granules. Am J Physiol Lung Cell Mol Physiol 2005; 288:L692-8.

26.Stamme C, Walsh E, Wright JR. Surfactant protein A differentially regulates IFN-gamma- and LPS-induced nitrite production by rat alveolar macrophages. Am J Respir Cell Mol Biol 2000; 23:772-9.

27.Brinker KG, Martin E, Borron P, Mostaghel E, Doyle C, Harding CV, et al. Surfactant protein D enhances bacterial antigen presentation by bone marrow-derived dendritic cells. Am J Physiol Lung Cell Mol Physiol 2001; 281:L1453-63.

28.Brinker KG, Garner H, Wright JR. Surfactant protein A modulates the differentiation of murine bone marrow-derived dendritic cells. Am J Physiol Lung Cell Mol Physiol 2003; 284:L232-41.

29.Lambrecht BN, Hammad H. Taking our breath away: dendritic cells in the pathogenesis of asthma. Nat Rev Immunol 2003; 3:994-1003.

30.Borron P, McCormack FX, Elhalwagi BM, Chroneos ZC, Lewis JF, Zhu S, et al. Surfactant protein A inhibits T cell proliferation via its collagen-like tail and a 210-kDa receptor. Am J Physiol 1998; 275:L679-86.

31.Borron PJ, Crouch EC, Lewis JF, Wright JR, Possmayer F, Fraher LJ. Recombinant rat surfactant-associated protein D inhibits human T lymphocyte proliferation and IL-2 production. J Immunol 1998; 161:4599-603.

32.Borron PJ, Mostaghel EA, Doyle C, Walsh ES, McHeyzer-Williams MG, Wright JR. Pulmonary surfactant proteins A and D directly suppress CD3+/CD4+ cell function: evidence for two shared mechanisms. J Immunol 2002; 169:5844-50.

33.Clark H, Palaniyar N, Strong P, Edmondson J, Hawgood S, Reid KB. Surfactant protein D reduces alveolar macrophage apoptosis in vivo. J Immunol 2002; 169:2892-9.

34.Palaniyar N, Clark H, Nadesalingam J, Shih MJ, Hawgood S, Reid KB. Innate immune collectin surfactant protein D enhances the clearance of DNA by macrophages and minimizes anti-DNA antibody generation. J Immunol 2005; 174:7352-8.
35.Madan T, Kishore U, Shah A, Eggleton P, Strong P, Wang JY, et al. Lung surfactant proteins A and D can inhibit specific IgE binding to the allergens of Aspergillus fumigatus and block allergen-induced histamine release from human basophils. Clin Exp Immunol 1997; 110:241-9.

36.Crouch E, Rust K, Veile R, Donis-Keller H, Grosso L. Genomic organization of human surfactant protein D (SP-D). SP-D is encoded on chromosome 10q22.2-23.1. J Biol Chem 1993; 268:2976-83.

37.Kolble K, Lu J, Mole SE, Kaluz S, Reid KB. Assignment of the human pulmonary surfactant protein D gene (SFTP4) to 10q22-q23 close to the surfactant protein A gene cluster. Genomics 1993; 17:294-8.

38.DiAngelo S, Lin Z, Wang G, Phillips S, Ramet M, Luo J, et al. Novel, non-radioactive, simple and multiplex PCR-cRFLP methods for genotyping human SP-A and SP-D marker alleles. Dis Markers 1999; 15:269-81.

39.Lahti M, Lofgren J, Marttila R, Renko M, Klaavuniemi T, Haataja R, et al. Surfactant protein D gene polymorphism associated with severe respiratory syncytial virus infection. Pediatr Res 2002; 51:696-9.

40.Floros J, Lin HM, Garcia A, Salazar MA, Guo X, DiAngelo S, et al. Surfactant protein genetic marker alleles identify a subgroup of tuberculosis in a Mexican population. J Infect Dis 2000; 182:1473-8.

41.Leth-Larsen R, Garred P, Jensenius H, Meschi J, Hartshorn K, Madsen J, et al. A common polymorphism in the SFTPD gene influences assembly, function, and concentration of surfactant protein D. J Immunol 2005; 174:1532-8.

42.Brown-Augsburger P, Hartshorn K, Chang D, Rust K, Fliszar C, Welgus HG, et al. Site-directed mutagenesis of Cys-15 and Cys-20 of pulmonary surfactant protein D. Expression of a trimeric protein with altered anti-viral properties. J Biol Chem 1996; 271:13724-30.

43.Heidinger K, Konig IR, Bohnert A, Kleinsteiber A, Hilgendorff A, Gortner L, et al. Polymorphisms in the human surfactant protein-D (SFTPD) gene: strong evidence that serum levels of surfactant protein-D (SP-D) are genetically influenced. Immunogenetics 2005; 57:1-7.

44.Turner MW. The role of mannose-binding lectin in health and disease. Mol Immunol 2003; 40:423-9.

45.Bochud PY, Hawn TR, Aderem A. Cutting edge: a Toll-like receptor 2 polymorphism that is associated with lepromatous leprosy is unable to mediate mycobacterial signaling. J Immunol 2003; 170:3451-4.

46.Kiechl S, Lorenz E, Reindl M, Wiedermann CJ, Oberhollenzer F, Bonora E, et al. Toll-like receptor 4 polymorphisms and atherogenesis. N Engl J Med 2002; 347:185-92.

47.Ehlen T, Dubeau L. Detection of ras point mutations by polymerase chain reaction using mutation-specific, inosine-containing oligonucleotide primers. Biochem Biophys Res Commun 1989; 160:441-7.

48.Green EK, Bain SC, Day PJ, Barnett AH, Charleson F, Jones AF, et al. Detection of human apolipoprotein E3, E2, and E4 genotypes by an allele-specific oligonucleotide-primed polymerase chain reaction assay: development and validation. Clin Chem 1991; 37:1263-8.

49.Pantelidis P, Lagan AL, Davies JC, Welsh KI, du Bois RM. A single round PCR method for genotyping human surfactant protein (SP)-A1, SP-A2 and SP-D gene alleles. Tissue Antigens 2003; 61:317-21.

50.Sorensen GL, Hjelmborg JB, Kyvik KO, Fenger M, Hoj A, Bendixen C, et al. Genetic and environmental influences of surfactant protein D serum levels. Am J Physiol Lung Cell Mol Physiol 2006; 290:L1010-7.

51.Tan CY, Chen YL, Wu LS, Liu CF, Chang WT, Wang JY. Association of CD14 promoter polymorphisms and soluble CD14 levels in mite allergen sensitization of children in Taiwan. J Hum Genet 2006; 51:59-67.
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top