跳到主要內容

臺灣博碩士論文加值系統

(44.210.21.70) 您好!臺灣時間:2022/08/11 16:47
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:康秀璠
研究生(外文):Hsiu-Fan Kang
論文名稱:探討剪應力對骨髓間質細胞之影響
論文名稱(外文):The effect of shear stress on stromal cells
指導教授:邱紫文邱紫文引用關係
指導教授(外文):Tzyy-Wen Chiou
學位類別:碩士
校院名稱:國立東華大學
系所名稱:生物技術研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:69
中文關鍵詞:剪應力
外文關鍵詞:shear stress
相關次數:
  • 被引用被引用:1
  • 點閱點閱:258
  • 評分評分:
  • 下載下載:25
  • 收藏至我的研究室書目清單書目收藏:0
骨髓間質細胞藉細胞激素之生產以及與造血細胞間之訊息傳遞、支持造血幹細胞/前驅細胞之增生與分化。本研究以AC6.21及HS-5兩株骨髓間質細胞株作為觀察剪應力影響骨髓間質細胞之材料,利用酵素連結免疫法,分析骨髓間質細胞之細胞激素生產動力學,並以二維蛋白質電泳配合基質輔助雷射脫附游離/飛行時間質譜儀之使用,探討細胞內蛋白質體表現之改變。
由實驗結果得知剪應力會影響骨髓間質細胞之細胞激素產量,並且對於不同種類之骨髓間質細胞有不同的影響。其中AC6.21能生產SCF、IL-6、GM-CSF及Flt-3,當受到10或20 dyne /cm2之剪應力作用後,其細胞激素表現量會下降。而HS-5能生產每毫升毫微克濃度 之IL-3、SCF、IL-6、IL-8及每毫升微微克濃度之GM-CSF,對HS-5施以適當剪應力能將IL-3生產量提升6倍,SCF生產量也可增加為4倍,而IL-8之生產量則提高5倍。
由結果得知,剪應力亦影響骨髓間質細胞內蛋白質體表現。AC6.21受到剪應力作用時約有1/3之蛋白質受到影響,其中70%有被調降的情形;而HS-5受到剪應力作用時約有1/2之蛋白質受到影響,其中80%有被調降的現象。
Bone marrow stromal cells support the proliferation and the differentiation of hematopoietic stem/progenitor cells through their cytokine production and the signal transduction mediated by their surface markers. In this study, two bone marrow stromal cell lines, AC6.21 and HS-5, were used to investigate the effect of shear stress on the stromal cells in a parallel-plate flow chamber. Under various shear stresses, the cytokine production of the cells was examined by the enzyme linked immunosorbant assay (ELISA), and the change in the intracellular protein expression was analyzed by the two-dimensional electrophoresis (2-DE) and the matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry.
The results indicated that the shear stress affected the cytokine production of both stromal cell lines in a different way. AC6.21 could produce SCF, IL-6, GM-CSF and Flt-3 and the production of these cytokines were reduced when the sear stress reached 10 or 20 dyne/cm2. The other stromal cell line, HS-5, produced IL-3, IL-6, IL-8 and SCF in the ng/ml level and GM-CSF in the pg/ml level. The concentrations of IL-3, SCF or IL-8 were increased to 6-, 4- or 5-fold higher respectively when the appropriate shear stresses were applied to the HS-5 cells.
Based on the results from 2-DE and MALDI-TOF /MS, it was shown that 1/3 of the total proteins expressed were affected by the shear stress and 70% of these affected proteins were down-regulated in AC6.21. In addition, about half of the proteins expressed were shown affected by the shear stress and 80% of these affected proteins were down-regulated in HS-5.
中文摘要-------------------------------------------------------------------------Ⅰ
英文摘要-------------------------------------------------------------------------Ⅱ
目錄-------------------------------------------------------------------------------Ⅲ
第一章 緒論----------------------------------------------------------------------1
第一節 研究目的----------------------------------------------------------------1
第二節 文獻回顧----------------------------------------------------------------2
1.2.1 幹細胞的活體外培養及增殖-------------------------------------------2
1.2.2 骨髓間質細胞-------------------------------------------------------------2
1.2.3 骨髓間質細胞於造血作用中所扮演的角色-------------------------5
1.2.4 剪應力對細胞的影響---------------------------------------------------10
1.2.5 蛋白質體學之研究背景----------------------------------------------13
1.2.5.1 二維式蛋白質電泳---------------------------------------------------13
1.2.5.2基質輔助雷射脫附游離/飛行時間質譜儀------------------------16

第二章 材料與方法-----------------------------------------------------------18
2.1 骨髓間質細胞培養-------------------------------------------------------18
2.1.1 AC6.21細胞之培養----------------------------------------------------18
2.2.2 HS-5(CRL-11882)細胞之培養---------------------------------------18
2.2剪應力----------------------------------------------------------------------19
2.2.1 剪應力對間質細胞生產細胞激素之影響-------------------------19
2.2.2剪應力對間質細胞細胞內蛋白質表現之影響--------------------21
2.2.2.1 AC6.21在不同剪應力環境下之培養-----------------------------21
2.2.2.2 HS-5在不同剪應力環境下之培養--------------------------------21
2.3 以酵素連結免疫法分析間質細胞之細胞激素表現---------------23
2.3.1 AC6.21生產細胞激素之分析-----------------------------------------23
2.3.2 HS-5生產細胞激素之分析--------------------------------------------23
2.4二維蛋白質電泳分析-----------------------------------------------------25
2.4.1 蛋白質樣品之製備-----------------------------------------------------25
2.4.1.1細胞外蛋白質樣品之製備------------------------------------------25
2.4.1.2細胞內蛋白質樣品之製備------------------------------------------25
2.4.2 一維等電焦集法(first-dimension isoelectric focusing)-----------27
2.4.2.1 再水合作用(rehydration) ------------------------------------------27
2.4.2.2電焦集法 (isoelectric focusing)-----------------------------------27
2.4.3 二維膠體電泳(second-dimension SDS-PAGE)-------------------27
2.4.3.1 IPG strip 平衡(equilibration)-------------------------------- ------28
2.4.4 SDS-PAGE---------------------------------------------------------------28
2.4.5染色-----------------------------------------------------------------------28
2.4.6膠體分析-----------------------------------------------------------------29
2.5蛋白質種類鑑別----------------------------------------------------------29
2.5.1 In gel digestion 及蛋白質萃取---------------------------------------30
2.5.2 MALDI-TOF分析------------------------------------------------------30

第三章 結果與討論----------------------------------------------------------32
3.1 剪應力對間質細胞生產細胞激素之影響-----------------------------32
3.1.1 剪應力對AC6.21生產細胞激素之影響----------------------------32
3.1.2 剪應力對HS-5生產細胞激素之影響-------------------------------37
3.2 剪應力對間質細胞胞內蛋白質表現之影響--------------------------43
3.2.1 剪應力對AC6.21細胞內蛋白質表現之影響----------------------43
3.2.2 剪應力對HS-5細胞內蛋白質表現之影響-------------------------48
3.2.3 蛋白質種類鑑定---------------------------------------------------------54

第四章 結論---------------------------------------------------------------------58

參考文獻-------------------------------------------------------------------------59
Aoyama K, Oritani K, Yokota T, Ishikawa J (1999) Stromal cell CD9 regulates differentiation of hematopoietic stem progenitor cells. Blood 93:2586-2594.

Barille S, Collett M, Thabard W, Bleunven C, Bataille R, Amiot M (2000) Soluble IL-6R alpha up-regulated IL-6, MMP-1 and MMP-2 secretion in bone marrow stromal cells. Cytokine 12: 1426-1429.
Bianco P, Riminucci M, Gronthos S, Robey PG (2001) Bone marrow stromal stem cells: nature, biology, and potential applications. Stem Cells 19: 180-192.

Bruder SP and Fox BS (1999) Tissue engineering of bone: Cell based strategies. Clinical Orthopaedics and Related Research. 367S: S68-S83.

Bjorn G, Gabriele R, Jiri V, Daniel R, Hanno L, and Urs J (2001) Proteomic analysis of the bacterial cell cycle. Proc. Natl. Acad. Sci. U. S. A. 98: 4681-4686.

Caplan AI and Bruder SP (2001) Mesenchymal stem cells:building blocks for molecular medicine in 21st century. Trends in Molecular Medicine 7: 259-264.

Chen N, Kao RH, and Lin CL (1999) Umbilical cord blood transplantation and cord blood banking. Tzu Chi Med J 11:301-309.

Civin CI (2000) Gene therapy in clinical applications. Stem Cells 18: 150-156.

Colter DC, Sekiya I, and Prockop DJ (2001) Identification of a subpopulation of rapidly self-renewing and multipotential adult stem cells in colonies of human marrow stromal cells. Proc. Natl. Acad. Sci. U. S. A. 98: 7841-7845.

Crawford ME, Cusik ME, Garrels JI (2000) Databases and knowledge resources for proteomics research. Proteomics: A trends guide, July 17-21.

Csar XF and Wilson NJ (2001) Proteomic analysis of macrophage differentiation. J. Biol. Chem. 276:26211–26217.

Eugene B (1993) Tissue engineering: Current perspectives. Birkhauser Inc., Boston, USA.

Hamblin AS (1993) Cytokines and cytokine receptors, 2nd edit. Oxford, New York, USA.

Kenllner R (2000) Promics. Concepts and perspectives. Fresenius J. Anal. Chem. 366: 517-524.

Koc EC and Burkhart WK (2000) A proteomics approach to the identification of mammalian mitochondrial small subunit ribosomal proteins. J. Biol. Chem. 275: 32585–32591.

Kudo SR and Saito MJ (2000) Shear-stress effect on mitochondrial membrane potential and albumin uptake in cultured endothelial cells. Biochem. and Biophy. Res. Commun. 270:616-621.

Kawada H, Ando K, Tsuji A, Shimakura Y (1999) Rapid ex vivo expansion of human umbilical cord hematopoietic progenitors using a novel culture system. Experimental Hematology 27: 904-915.

Lu L, Shen RN and Broxmeyer HE (1996) Stem cells from bone marrow, umbilical cord blood and peripheral blood for clinical application: Current status and future application. Critical Reviews in Oncology/Hematology 22: 61-78.

Mayani H and Lansdorp PM (1998) Biology of human umbilical cord blood-derived hematopoietic stem/progenitor cells. Stem Cells 16: 153-165.

McAdams TA, Winter JN, Miller WM, and Papoutsakis ET (1996) Hematopoietic cell culture therapies (part II): Clinical aspects and applications. Tibtech 14: 388-396.

Mellado DN, Rodríguez JM, Carmona M, Gonzá J and Prito J (1999) Ex vivo expansion and maturation of CD34-positive hematopoietic progenitors optimization of culture conditions. Leukemia Res 23: 1035-1040.

Morgan JR and Yamush ML (1999) Tissue engineering methods and protocols. Humana Press Inc, USA.

Naour FL, Hoenkirk L, Annabelle G, David EM, Pascal L, James DG, Samir H, and Laura B (2001) Profiling changes in gene expression during differentiation and maturation of monocyte-derived dendritic cells using both oligonucleotide microarrays and proteomics. J. Biol. Chem. 276: 17920-17931.

Robledo MM and Teixido J (1997) TGF-beta 1-binding proteins on human bone marrow stromal cells. Leuk Lymphoma 27: 509-515.

Roeckline BA and Storb BT (1995) Functionally distinct human marrow stromal cell lines immortalized by transduction with human papilloma virus E6/E7 genes. Blood 85: 997-1005.

Sakai KM and Mohtai YI (1998) Fluid shear stress increases transforming growth factor beta 1 expression in human osteoblast-like cells: Modulation by cation channel blockades. Calcified Tissue International 63: 515-520.

Shih CC, Mickey CT. Hu, Hu J, Medeiros J and Forman SJ (1999) Long-term ex vivo maintenance and expansion of transplantable human hematopoietic stem cells. Blood 94: 1623-1636.

Shih CC, Digiusto D and Forman SJ (2000a) Ex vivo expansion of transplantable human hematopoietic stem cells: Where do we stand in the year 2000? J. Hematotherapy and Stem Cell Res. 9:621-628.

Shih CC, M CT Hu, Hu J, Weng Y, Yazaki P J, Medeiros J, and Forman SJ (2000b) A secreted and LIF-mediated stromal cell-derived activity that promotes ex vivo expansion of human hematopoietic stem cells. Blood 95: 1957-1966.

Shimakura Y, Kawada H, Ando K, Sato T, Nakamura Y, Tsuji T, Kato S, and Hotta T (2000) Murine stromal cell line HESS-5 maintains reconstituting ability of ex vivo-generated hematopoietic stem cells from human bone marrow and cytokine-mobilized peripheral blood. Stem Cells 18: 183-189.
Theze J (1999) The cytokine network and immune function. Oxford University Press. New York, USA.

Tony SZ Dung, Lan WC and Yang SF (1999) Osteogenic characterization of bone marrow stromal cells. Chin. Dent. J. 18: 175-190.

Vido KD, Spector GL, S. Lopez MB, Toledano JL (2001) A proteome analysis of the cadmium response in Saccharomyces cerevisiae. J. Biol. Chem. 276: 8469–8474.

Xu Q, Scheet G, Li C, Hu Y and Wick G (2000) Mechanical stress-induced heat shock protein 70 expression in vascular smooth muscle cells is regulated by Rac and Ras small G proteins but not mitogen-activated protein kinase. Circulation Research: Journal of the American Heart Association 86: 1122-128.

Yoshio T, Fumiko S, Shuhei K Shuji Y, Hiroko T, Risa K and Joji A (1994) Fluid shear stress increases the expression of thrombomodulin by cultured human endothelial cells. Biochem. and Biophys. Res. Commun. 205: 1345-1352 .

Zandstra PW, Conneally E, Petzer AL, Piret JM and Eaves CJ (1997) Cytokine manipulation of primitive human hematopoietic cell self-renewal. Proc. Natl. Acad. Sci. U. S. A. 94: 4698-4703.

Zhang HT, Janet EK, Kevin M, Mark IG and James E (2001) Protein quantification from complex protein mixtures using a proteomics methodology with single-cell resolution. Proc. Natl. Acad. Sci. U. S. A. 98: 5497-5502.
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top
1. 莊文星、陳汝勤(1989) 臺灣北部火山岩之定年與地球化學研究,經濟部中央地質調查所彙刊,第五號,第31-66頁。
2. 鐘樹椽(民85)。互動性在電腦輔助教學設計上之必要性。教學科技與媒體,12,15-20。
3. 鐘樹椽(民82)。電腦合作學習效果之探究。教師之友,34(1),8-12。
4. 蘇婉容(民83)。圖片的應用有助於智障兒童學習認字嗎?國小特殊教育,16,57-59。
5. 蔣德勉(民72)。電腦輔助教學的發展。科學教育月刊,64,25-31。
6. 鄭晉昌(民82):自「情境學習」的認知觀點探討電腦輔助教學中教材內容的設計-從幾個學科教學系統談起。教學科技與媒體,12,3-14。
7. 楊憲明(民87)。障礙學生文字辨識自動化處理之分析研究。特殊教育與復健學報,6,15-37。
8. 游惠美、孟瑛如(民87)。電腦輔助教學應用方式對國小低成就兒童注音符號補救教學成效之探討。特殊教育與復健學報,6,307-347。
9. 陳麗玲(民81)。閱讀技能發展及補救教學策略。國教輔導,289,35-39。
10. 陳淑絹(民85)。由國民小學「閱讀指導現況調查」談閱讀策略教學的可行性。國教輔導,36(2),24-29。
11. 郭生玉(民75):單一受試(小N)的實驗設計。中國測驗學會測驗年刊,33,143-162。
12. 唐存慧(民77)。電腦輔助教學之發展與研究。國教世紀,24(3),32-35。
13. 施婉悅(民85)。電腦輔助教學與國小高年級學童數學科學習適應及數學態度之相關研究。臺南師院學生學刊,17,1-12。
14. 邱貴發、鍾邦友(民82)。情境學習理論與電腦輔助學習軟體設計。臺灣教育,510,23-29。
15. 林素貞(民87):相似字與非相似字呈現方式對國小一年級國語科低成就學生生字學習效困之比較。特殊教育與復健學報,6,261-277。