臺灣博碩士論文加值系統

腫瘤內皮標誌物一(TEM1) 是丙型凝集素跨膜受體家族中的第一型穿膜糖蛋白，主要表達於間質幹細胞、平滑肌細胞、周細胞以及纖維母細胞。TEM1可能參與肝臟、腎臟以及肺臟等器官纖維化的發病機制。已知乙型轉化生長因子（TGF-β）參與纖維細胞的活化及膠原蛋白合成的增加是組織纖維化的關鍵。因此推測TEM1可能參與TGF-β所介導的訊息傳遞進而造成纖維化。當使用小分子干擾核醣核酸(siRNA)去抑制人類真皮纖維母細胞(NHDF)表達TEM1，可見smad2被TGF-β誘導入核的情況下降。通過蔗糖梯度離心分離NHDF中的脂質和非脂質筏區域後，發現TEM1和第二型乙型轉化生長因子受器(TβRII)在這兩個區域都有分布。另外，TEM1的減少不會影響TβRII的分布位置。實驗發現，TEM1和TβRII在NHDF亞胞器產生共位現象。通過共免疫沉澱進一步證明TEM1與TβRII相連。此外，TEM1不是與網格蛋白輕鏈(clathrin light chain)共位，而是與一號小窩蛋白(caveolin-1)產生共位。另一方面，當限制表達TEM1時，caveolin-1的蛋白表現些微上升，第一、第二型乙型轉化生長因子受器的蛋白表現量卻下降。在博萊黴素誘導皮膚纖維化的小鼠模型中，TEM1缺失的小鼠真皮厚度變薄且膠原沉積變少。此外在臨床方面，在增生性疤痕的組織中，可發現TEM1被顯著表達，且與TβRII共位。綜合以上，TEM1可能在真皮纖維化中透過與TβRII相互作用而抑制乙型轉化生長因子所介導的訊息傳導。

Tumor endothelial marker 1 (TEM1) is a type I transmembrane glycoprotein belonging to the “Group XIV”, a family of C-type lectin transmembrane receptors. Expression of TEM1 was found in mesenchymal stem cells, smooth muscle cells, pericytes, and fibroblasts. TEM1 was shown to be involved in the pathogenesis of fibrotic diseases in liver, kidney, and pulmonary fibrosis. Transforming growth factor-β (TGF-β) plays a key role in the tissue fibrosis by modulating the activation of fibroblasts and increasing collagen synthesis. We proposed that TEM1 might participate in TGF-β-mediated signaling. TGF-β-mediated signaling pathways and downstream protein expressions in normal human dermal fibroblasts (NHDFs) were inhibited by transfection with small interfering RNA of TEM1. Decreased TGF-β-induced nuclear translocation of Smad2 was found in TEM1 knockdown NHDFs. Both TEM1 and TGF-β receptor II (TβRII) were found in the lipid and non-lipid raft fractions in NHDFs by sucrose gradient centrifugation. Besides, knocking down TEM1 did not affect the distribution of TβRII. Interestingly, TEM1 and TβRII were co-localized in subcellular organelles in fibroblasts. The association of TEM1 and TβRII was demonstrated by co-immunoprecipitation. In addition, the co-localization of TEM1 with caveolin-1 but not clathrin light chain was observed in NHDFs. Silencing TEM1 in NHDFs increased the protein levels of caveolin-1 and reduced the protein expressions of TGF-β receptor I and II. TEM1 knockout exhibited thinner thickness and lower collagen deposition in the dermis in the mouse model of bleomycin-induced dermal fibrosis. In clinical samples, TEM1 expression was significantly up-regulated and co-localized with TβRII in hypertrophic scar. Taken together, TEM1 plays a critical role in dermal fibrosis by interacting with TβRII and suppressing TGF-β-mediated signaling.

中文摘要 ...............Ⅰ
Abstract ...............Ⅱ
Acknowledgement ............Ⅲ
Contents ...............Ⅳ
Abbreviation ..............Ⅶ
Instruments .............Ⅸ
Reagents and Chemicals ...........XI
Introduction ...............1
I. Physiological Functions of Dermal Fibroblast. ......1
II. The Mechanism of Fibrosis. .........2
III. The TGF-β-Mediated Signaling Pathway. ......3
IV. Endocytic Regulation of TGF-β Signaling. ........3
V. Physiological Function of Tumor Endothelial Marker 1. .....5
Specific Aim ..............7
Materials and Methods ............8
I. Cell Culture .............8
II. Cell Counting .............9
III. Cell Freezing ............10
IV. Cell Thawing ............11
V. siRNA teansfection..........12
VI. Time Course or Signal Starvation Assay .......13
VII. Protein degradation Assay ..........14
VIII. Scratch Assay ............16
IX. Co-Immunoprecipitation Assay...16
X. Density Gradient Ultracentrifugation....18
XI. Western Blot Analysis............21
XII. Immunocytochemical Staining.........24
XIII. Real-Time Polymerase Chain Reaction........26
XIV. Hematoxylin and Eosin Staining..........28
XV. Sirius Red Staining...30
XVI. Immunohistochemistry Staining........32
XVII. Animal model of bleomycin-induced dermal fibrosis....34
Results..............35
I. TEM1 knockdown inhibits TGF-β-induced signaling in NHDFs. ...35
II. TEM1 is associated and co-localized with TGF-β receptor II in NHDFs. .36
III. TEM1 and TGF-β receptor II are located in intracellular vesicles. ..36
IV. TGF-β receptor is significantly diminished in TEM1 knockdown NHDFs. .37
V. Animal model of bleomycin-induced dermal fibrosis......38
VI. Bleomycin-induced dermal fibrosis was reduced in TEM1 gene deleted mouse. ..............39
VII. TEM1 is associated with TGF-β receptor II in hypertrophic scar. ...39
Conclusion..............40
Discussion...............41
Figures..............45
1. TEM1 knockdown inhibits TGF-β-mediated signal in NHDFs....45
2. TEM1 silencing down-regulate the translocation of Smad2 into nuclear in NHDFs. ..............46
3. TEM1 is associated and co-localized with TβRII in NHDFs. .47
4. TEM1 and TβRII are located in intracellular vesicles. ....48
5. TGF-β1 changes the co-localization of TEM1 with caveolin-1 in NHDFs. ..49
6. TGF-β receptor is significantly diminished in TEM1 knockdown NHDFs..50
7. TEM1 knockdown facilitates the degradation of TGF-β receptor in NHDFs. ..51
8. Animal model of bleomycin-induced dermal fibrosis. ....52
9. TEM1 is up-regulated in dermal fibrosis of mice. .......53
10. Bleomycin-induced dermal fibrosis was reduced in TEM1 gene deleted mouse. ..............54
11. TEM1 is associated with TGF-β receptor II in hypertrophic scar. ...55
12. Schematic model is proposed for the biological function of TEM1 in TGF-β-mediated signal transduction...56
13. The effect of TEM1 on TGF-β receptor expression in NHDFs after TGF-β1 treatment.....57
References...............58

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