臺灣博碩士論文加值系統

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

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