| Summary: | 碩士 === 中國醫藥大學 === 醫學檢驗生物技術學系碩士班 === 97 === Thrombomodulin (TM) is an endothelial cell surface glycoprotein that performs anticoagulant, cell-cell adhesion, and anti-inflammatory functions in various tissues. TM has been detected in platelets, megakaryocytes, monocytes, neutrophils, smooth muscle cells, it is also expressed in epithelial keratinocyte. But the function of thrombomodulin in epithelial tissue remains unclear. In this thesis, TM deficiency HaCaT (HaCaT-siTM) cell line was used to study the biological effects of TM on epithelial tissue. We found that TM regulated the cell morphology, proliferation and migration in HaCaT cells. Altered protein expressions between HaCaT keratinocytes and TM deficiency HaCaT (siTM) cells were identified by 2D-DIGE (2-D Fluorescence Difference Gel Electrophoresis) coupled with mass spectrometry. Difference gel electrophoresis (DIGE) is a form of gel electrophoresis where up to three different protein samples can be labeled with fluorescent dyes (Cy3, Cy5, Cy2) prior to two-dimensional electrophoresis. Total 1100 spots wae detected by typhoon 9200. Total of 25 proteins were significantly (P < 0.05,ratio＞1.5) changed in abundance between HaCaT and HaCaT-siTM cells. Seventeen proteins were successfully identified by MALDI-TOF/TOF. We showed that knock down TM expression could lead to the increased expression of RAD23 homolog B, elongation factor 1-delta, dUTPase, and proteasome subunit alpha type-1, whereas Annexin A3 and procathepsin D were being down-regulated the expression of. Annexin A3 and procathepsin D were further confirmed by Western blotting. In conclusion that TM could lead to keratinocytes transformation but the role of annexin A3 and procathepsin D in this process remains to be further studied.
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