凝血酶調節素(Thrombomodulin, TM)是一種表現於血管內皮細胞膜表面的醣蛋白，其主要功能是參與血液抗凝集反應。凝血酶調節素除分布於內皮細胞外，於其它組織細胞如：胎盤融合滋胚層、單核球、嗜中性白血球、上皮細胞、平滑肌細胞、角質細胞、黏液束細胞、腦脊膜細胞及腫瘤細胞也都有TM的表現。在老鼠胚胎發育中，TM的分布在胚胎外的胎盤細胞、心血管發育系統、上皮細胞、軟骨以及腦的部分區域，其分佈如此廣泛，顯示出TM除了參與抗凝血的角色外，可能還有其它功能。
為了針對TM的功能做進一步研究，本論文利用酵母菌表現系統，表現了不同TM片段的重組蛋白質，包括TMD123、TMD12、TMD23、TMD2、TMEGF1-3、TMEGF1-4、TMEGF1-5，並於TM重組蛋白與c-myc融合蛋白(fusion protein)間加入了腸激酶切割序列(AspAspAspAspLys)，利用含Zeocin的培養基篩選出含有載體基因的XL-33酵母菌細胞株，利用小量表現挑出表現量高的菌株，結果顯示所有TM蛋白片段皆能成功表現於酵母菌系統。
將TMD123發酵槽培養液進一步純化時發現，當加入鎂離子後，似乎可緩解TMD123蛋白片段分解速率；當外加EDTA或鈣離子存在下，無法減緩其分解速率。為何TMD123無法穩定存在於發酵液中，或於鎂離子存在下，能減緩其分解速率，仍需進一步實驗來確認。
不同於TMD123蛋白片段，TMD23是較穩定的蛋白片段，經由DEAE 陰離子交換樹脂以及親合性鎳離子螯合樹脂的純化後，即可得到高純度的TMD23蛋白，經過N端蛋白質定序後確認為正確表現片段。進行protein C活化試驗發現純化後的TMD23具有活化protein C的功能，進一步將此純化後的TMD23蛋白與BSA進行FITC標定後，分別與細胞株BAEC、bPAEC、HaCaT、A549作用4小時後，在螢光顯微鏡下觀察，發現TMD23被BAECs吞噬的現象較明顯。將TMD23-FITC與BAECs作用不同時間後，分別觀察BAEC吞噬TMD23的現象，於共軛焦顯微鏡觀察下，發現TMD23蛋白會於30分鐘內與細胞的細胞膜結合，接著TMD23蛋白會在細胞膜上聚集(capping)，再吞噬進入細胞內。

Thrombomodulin (TM), a glycoprotein found on the endothelial cell surface, plays an important role in regulation of blood coagulation. It was also demonstrated that TM was widely distributed in placental syncytiotrophoblasts, monocytes, neutrophils, epithelial cells, smooth muscle cells, keratinocytes, synovial lining cells, meningeal cells, and tumor cells. During the mouse development, TM is expressed in extraembryonic placental tissues, in the developing cardiovascular system, airway epithelia, cartilage, and in restricted area of the brain. This raises the possibility that TM possesses functions distinct from those related to hemostatic regulation.
In order to study the functions of extracellular domains of TM, we constructed plasmid containing DNA of TM domains including：TMD123, TMD23, TMD12, TMD2, TMEGF1-3, TMEGF1-4, and TMEGF1-5.The DNA constructs were subcloned into pPICZA expression vector containing c-myc fusion peptide and enterokinase cutting site (AspAspAspAspLys). These constructs were transfected into X-33 and the highly expressed clones were selected by Zeocin. All the recombinant proteins were successfully expressed and secreted into the medium in Pichia expression system.
TMD123 protein was expressed in large-scale bioreactor. TMD123 protein was very unstable and tends to decompose during purification. Mg2+ could slow down the TMD123 decomposition. EDTA and Ca2+ had no effect to improve the stability of TMD123. Why TMD123 was unstable in fermentor medium and the protective effect of Mg2+ still needs further investigation.
TMD23 protein was large-scale expressed by bioreactor system and purified by DEAE -Sepharose and Ni2+-chelating Sepharose and the recombinant TMD23 was analyzed by SDS/PAGE and Western blotting. Purified TMD23 showed a sharp band on SDS/PAGE. The purified TMD23 was labeled by FITC for study the binding and internalization with the BAECs, bPAECs, HaCaT cells ,or A549 cells for 4 h at 37℃. The FITC- labeled TMD23 could be internalized into the BAECs faster than other cells. Using confocal microscope, we found that the FITC -labeled TMD23 interacted with the cell membrane; the protein aggregated and engulfed into the BAECs within 30 minutes.

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