臺灣博碩士論文加值系統

結締組織生長因子(CTGF)為CCN(Connective tissue growth factor, Cysteine-rich 61, Nephroblastoma overexpressed)家族的成員，其具有的生物作用相當廣泛，包括細胞增生分裂、產生細胞外間質蛋白、血管生成及抑制生長等等。在組織纖維化的過程中，結締組織生長因子受到乙型轉型生長因子(TGF-β)的刺激而表現量增加。因此為瞭解重組的人類結締組織生長因子(rhCTGF)是否會影響HK2(人類近端腎小管)細胞的生長及細胞外間質蛋白的表現，我們將不同濃度的重組人類結締組織生長因子加到培養的HK2細胞，另一方面使結締組織生長因子在HK2細胞中表現來檢視。我們從HK2細胞中萃取total RNA並作為模版，以特異的CTGF引子進行coupled one-step reverse transcription PCR獲得CTGF基因，將選殖出的基因接上TA載體，以ABI3730定序系統進行DNA序列的定序後，利用BLAST比對找出正確的基因序列。正確的基因分別接合至pET28a(原核表現載體)及pcDNA3.1(哺乳類表現載體)，其一的pET28a/CTGF質體送入BL21(DE3)大腸桿菌中，在IPTG誘導下使重組人類結締組織生長因子(rhCTGF)大量表現，並以Ni2+吸收層析法純化此蛋白。pcDNA3.1/CTGF以lipofetAMINE方式送入HK2細胞中表現CTGF protein。三天後以MTT方法及3H-proline併入法評估HK2細胞的生長及細胞外間質蛋白的產生有否受到外加的CTGF蛋白及細胞中表現的CTGF而影響。外加40 ng/ml的重組人類結締組織生長因子(rhCTGF)到HK2細胞及細胞內結締組織生長因子的過表現均明顯地抑制HK2細胞的增生，而膠原蛋白產生量隨著重組結締組織生長因子濃度增加有上升趨勢。因此，我們純化的重組人類結締組織生長因子(rhCTGF)具有生物活性且結締組織生長因子在HK2細胞中抑制細胞生長及促進細胞外間質蛋白產生。

Connective tissue growth factor (CTGF) is a member of the CCN (Connective tissue growth factor, Cysteine-rich 61, Nephroblastoma overexpressed) family of proteins, which exhibits diverse biological actions depending on the cell types including: mitogenesis, extracellular matrix production, angiogenesis and growth inhibition, etc. CTGF was stimulated by transforming growth factor-β and overexpressed during tissue fibrosis. Thus, to determine whether rhCTGF influences the growth of HK2(human proximal tubular) cells and production of extra-cellular matrix, we exposed HK2 cells to rhCTGF and transiently overexpressed CTGF in HK2 cell. Complete open reading frame of CTGF gene was obtained by coupled one-step RT PCR reactions using CTGF specific primers on mRNA population isolated from HK2 cells. PCR products were subcloned into the TA vector. Sequencing was performed using an automated ABI 3730 DNA sequencing system. The sequences obtained were compared using BLAST searches. Resulting fragments were inserted into the pET28a (prokaryotic expression vector) and pcDNA3.1 (mammalian expression vector) separately. Recombinant human CTGF protein was overexpressed under IPTG induction after introduced into BL21 (DE3), then was purified using Ni2+ absorption chromatography under denaturing condition. The constructed pcDNA3.1/CTGF vector driven by a cytomegalovirus promoter was transiently transfected into HK2 cell by use of LipofetAMINE (Invitrogen). The effects of CTGF and TGF-β on HK2 or CTGF overexpression on HK2 cells in terms of cell growth and ECM production were evaluated by MTT assay and 3H-proline incorporation. We found that CTGF(40 ng/ml) and CTGF overexpression significantly inhibited cell proliferation. Recombinant CTGF also dose-dependently induced collagen production. Thus, we have produced recombinant human CTGF protein which has biological activity and CTGF is a growth inhibitor, which increased ECM production in HK2 cells.

中文摘要 2
英文摘要 3
緒論 4
研究目的 17
實驗材料 18
實驗方法 20
結果 33
討論 37
結論 47
參考文獻 49
圖 60
附錄 71

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