臺灣博碩士論文加值系統

結締組織生長因子於人類滑液膜細胞扮演之生理角色

中文摘要
目的:探討人類結締組織生長因子(CTGF)對於人類滑液膜細胞(synoviocytes)之生理影響。方法：將人類滑液膜組織做初代細胞培養，處理結締組織生長因子蛋白(100 ng/mL)，觀察結締組織生長因子對於人類滑液膜細胞增生(proliferation)、移行(migration)及分化(differentiation)之影響。本論文利用反轉錄聚合酶鏈鎖反應(RT-PCR)觀察細胞在骨形態蛋白家族(BMP family)之表現；使用西方墨點法(Western blot)探討滑液膜細胞於骨形態蛋白2(BMP2) 之蛋白表現，與mitogen-activated protein kinase (MAPK)、PI3 kinase(PI3K) 之磷酸化訊息傳遞途徑。結果：初代培養人類滑液膜細胞處理rCTGF後，細胞增生能力較對照組無顯著增加，同時也不具有時間效應關係。在細胞移行方面，滑液膜細胞加入rCTGF後，細胞移動能力並無改變。但在分化方面，我們發現rCTGF能誘導滑液膜細胞分化成硬骨，而不會促進軟骨及脂肪之分化。在下游訊息方面，rCTGF處理滑液膜細胞於骨形成蛋白2之蛋白與mRNA表現隨時間呈現有意義的上升。MAPK訊息傳遞路徑中，p38磷酸化蛋白明顯增加，而JNK 磷酸化蛋白有減弱之趨勢。結論：在細胞實驗證明，CTGF無促進滑液膜細胞增生及移動的能力，但具有促進硬骨分化之能力，且CTGF誘導滑液膜細胞之硬骨分化作用可能是經由MAPK/p38這條細胞內訊息傳遞途徑。未來可發展CTGF為臨床上促進關節內硬骨生成之蛋白質分子藥品。

關鍵詞：結締組織生長因子、滑液膜細胞、分化、骨形態蛋白2、硬骨生成

The Physiological Roles of Connective Tissue
Growth Factor in Human Primary Synoviocytes

Abstract
Purpose: The physiological roles of connective tissue growth factor (CTGF) in human primary synoviocytes. Methods: Human synovial tissue was primary cultured, and treated with rCTGF (100 ng/mL). Here we investigated the function of CTGF in proliferation, migration, and differentiation on human synoviocyte. Bone morphogenetic protein family was examined by RT- PCR and Western blotting assay. To exam the signal transduction pathway, phosphorylation status of mitogen-activated protein kinase (MAPK) and PI3 kinase (PI3K) proteins would be checked after CTGF treatment with Western blots. Results: We found that CTGF had no significant time- or dose- dependent effects in cell proliferation and migration in human synoviocytes. However, rCTGF treatment showed ability to differentiate into osteogenic cells, but not chondrogenic or adipogenic cells after indicated times. To screen the BMP family, the data demonstrated that rCTGF increased BMP2 mRNA and protein expression. In signal transduction, phosphorylated MAPK/p38 markly increased after rCTGF treatment, but phosphorylated JNK was decreased. Conclusion: Our data demonstrated that CTGF had no effect in proliferation and migration of human synoviocytes, but could promote osteogenesis differentiation. We suggested that CTGF-induced osteogenesis differentiation possibility by MAPK/p38 signaling pathway. In perspective, CTGF is a potent molecular protein drug to induce osteogenesis in articular disease.

Key words: CTGF, synoviacytes, differentiation, BMP2, osteogenesis

目錄

原創聲明書............................................... ii
學位考試審定書........................................... iii
中文摘要.................................................iv
英文摘要.................................................vi
謝誌.....................................................viii
目錄.....................................................x
表目錄...................................................xii
圖目錄...................................................xiii

第壹章　緒論
第一節　研究背景....................................1
第二節　研究目的....................................5
第三節　研究之重要性................................5

第貳章　文獻探討
第一節　結締組織生長因子............................6
第二節　滑液膜......................................9
第三節　分化........................................11

第參章　研究方法與材料
第一節　初代細胞培養................................13
第二節　細胞複製分析................................15
第三節　細胞移行分析................................15
第四節 西方墨點法..................................16
第五節 核糖核酸抽取................................18
第六節 反轉錄¬¬-聚合酶鏈鎖反應.......................19
第七節 鹼性磷酸酶組織染色...........................20
第八節 Alcian Blue組織染色..........................22
第九節 脂肪滴染色...................................23
第十節 統計分析.....................................24

第肆章　結果與討論
第一節　人類滑液膜細胞培養之建立.......................25
第二節　結締組織生長因子對人類滑液膜細胞增生之影響.......26
第三節　結締組織生長因子對人類滑液膜細胞移行之影響.......27
第四節 結締組織生長因子對人類滑液膜細胞分化之影響.......28
第五節 人類滑液膜細胞處理結締組織生長因子之骨形態蛋白
mRNA表現.....................................32
第六節 人類滑液膜細胞處理結締組織生長因子之訊息傳
遞.... ......................................34
第七節 結締組織生長因子對人類滑液膜細胞之生理功能........36
第八節 滑液膜細胞與分化...............................37
第九節 滑液膜細胞與骨形態蛋白..........................38

第伍章　結論與建議
第一節　結論.........................................39
第二節　建議.........................................40

參考文獻　
外文部分.............................................41

附錄　
附錄一 研究計畫審查證明書..............................48

表目錄

表1 西方墨點法使用之一級抗體來源及條件...........................46
表2 反轉錄聚合酶鏈鎖反應實驗中使用之引子及條件....................47

圖目錄

圖1 直接切割培養出之人類滑液膜細胞 ...........................13
圖2 酵素分解方法取得之人類滑液膜細胞...........................14
圖3 MG63細胞ALP染色後呈現骨新生之籃紫色標記....................21
圖4 由人類滑液膜組織培養出之滑液膜細胞.........................25
圖5 結締組織生長因子對人類滑液膜細胞增生之影響..................26
圖6 結締組織生長因子對人類滑液膜細胞移行之影響..................27
圖7 人類滑液膜細胞處理結締組織生長因子之分化表現.................29
圖8 人類滑液膜細胞處理CTGF在-SMA、COL-1之mRNA表現............31
圖9 人類滑液膜細胞處理CTGF在BMP家族之mRNA表現..................33
圖10 人類滑液膜細胞處理結締組織生長因子在BMP2、Sox9、
及訊息傳遞路徑MAPK及PI3K之蛋白表現........................34
圖11 結締組織生長因子誘導滑液膜細胞之硬骨分化可能路徑.............39

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