論文名稱:

口腔癌細胞株核內類胰島素生長因子結合蛋白-5之表現與功能

 

The Expression and Function of Nuclear Insulin-like Growth Factor Binding Protein-5 (IGFBP-5) in an Oral Cancer Cell Line

研究生:

張舜閔  Shun-Min Chang

 

(以作者名查詢陽明大學館藏系統)

 

(以作者名查詢全國圖書書目資訊網)

指導教授:

林姝君  Shu-Chun Lin

        學位類別:

碩士

        學校名稱:

國立陽明大學

系所名稱:

口腔生物研究所

            學號:

39217003

          學年度:

93

          語文別:

中文

          出版年:

94

關鍵字:

類胰島素生長因子  IGF

 

類胰島素生長因子結合蛋白-5  IGFBP-5

 

基質金屬蛋白酶  MMP

全文說明:

(本論文 20070901 對校內公開)

 

電子全文

        論文頁數:

90

摘要:

IGFBPIGF之重要結合蛋白,近年來有學者指出IGFBP-5除了可與IGF結合並影響IGF的生化功能外,本身也可以不依賴IGF而促使細胞移動,IGFBP-5更可以進一步進入到細胞核中但功能不明,IGFBP-5蛋白質序列的N端還具有活化轉錄的功能,但其調控基因亦不為所知。實驗室先前的研究發現在口腔癌化及大部分口腔鱗狀細胞癌細胞株內,IGFBP-5的表現有極明顯的下降;且利用外加的IGFBP-5加入不表現IGFBP-5的腫瘤細胞株中,可以抑制腫瘤細胞生長,顯示IGFBP-5之調降與口腔癌化之密切關係;口腔癌組織基質金屬蛋白酶(matrix metalloproteinasesMMPs)與侵襲有關且有大量的表現,且實驗室先前也觀察到有MMP-13的表現在癌組織的細胞核中。在本實驗中藉質體將IGFBP-5-EGFP融合蛋白在細胞內大量表現,篩選穩定表現的細胞株,經由共軛焦顯微鏡觀察證實於細胞核中有大量代表IGFBP-5綠螢光的表現,且在免疫細胞染色中也看到相同結果,這些細胞株,不論在mRNA或是蛋白質的層面皆有大量IGFBP-5-EGFP表現,並且此IGFBP-5-EGFP可在細胞外偵測到,此種融合蛋白也具有與IGF-1結合的能力,而表現此融合蛋白的細胞株的族群倍增時間延長5小時,細胞的移動能力也有提升。在酵素切割的實驗中,首次發現MMP-7MMP-13可以將IGFBP-5當成受質,隨著作用時間的增加將IGFBP-5切割成小片段,當加入MMP的抑制劑時確實可以抑制MMP-7MMP-13的切割能力,證明MMP具降解IGFBP-5的作用,而IGFBP-5-EGFP融合蛋白亦受MMP-7MMP-13切割,本研究建立之過度表現系統可進一步探討IGFBP-5對口腔癌表型之影響。

 

Insulin-like growth factor binding protein 5 (IGFBP-5) is an important IGF binding protein. It can exert regulatory functions when binding with IGF. It also has IGF-independent function in regulating cell mobility. Recent studies have indicated that IGFBP-5 can enter nucleus with unclear function. N-terminal sequence of IGFBP-5 was postulated have transcription-activating function, but the target genes are unknown. In our laboratory, we have identified that IGFBP-5 was down-regulated in the vast majority of oral squamous cell carcinoma (OSCC) and OSCC cell lines. Treatment with recombinant IGFBP-5 remarkably inhibited the growth of OSCC cells without IGFBP-5 expression. Down-regulation of IGFBP-5 seems to be contributive for OSCC growth. Matrix metalloproteinases (MMPs) is a group of preoteinases associated with tumor invasion, that are frequently overexpressed in cancers. Our previous study has specified the presence of MMP-13 in cell nuclei of OSCC. This study established stable cells line transfected with plasmid expressing IGFBP-5-EGFP fusion protein. Both immunofluoresence resolved by confocal microscopy and immunocytochemistry showed eminent nuclear-localization of IGFBP-5-EGFP in these cells. Abundant mRNA and protein expression was detected in nuclear extract and whole cell lysate. Besides, this fusion protein can be detected in supernatant. This fusion protein exhibits binding affinity to IGF-1. These stable cells had extended population-doubling time 5 h longer than parental cells. They also exhibited increase in cellular mobility. In enzyme digestion experiments, we identified for the first time that MMP-7 and MMP-13 can cleave recombinant IGFBP-5 in a time-dependent manner. MMP inhibitor drastically hindered such cleavage. Interestingly, IGFBP-5-EGFP fusion protein is also a substrate of MMP-7 and MMP-13. This study established an overexpression system that can be used to approach the phenotypic impacts of IGFBP-5 on OSCC.