Endocan促進內皮細胞轉移與侵犯能力之研究

Endocan Promotes the Migration and Invasion Ability of Endothelial Cells

周書帆(Shu-Fan Chou)

指導教授 ： 王萬波

國立臺灣大學/醫學院/微生物學研究所/碩士(2007年)

https://doi.org/10.6342/NTU.2007.10413

摘要

Endocan(又名內皮細胞特異表現分子，Esm-1)為一分子量約50 kDa的可溶性蛋白多醣，由核心蛋白與一條condroitin/ dermatan sulfate（CS/DS）醣基鏈組成，主要表現於人體肺、腎組織及內皮細胞中。研究顯示，內皮細胞的endocan會受到週邊環境因子的調控；如發炎時大量表現的細胞激素TNF-α，及促進細胞增生、血管新生的生長因子VEGF-A等。前人之in vitro實驗證實，endocan可與肝細胞生長因子HGF/SF共同作用，藉其醣基鏈加強HGF/SF促進細胞增生的能力。文獻指出，於老鼠體內移植穩定表現endocan之HEK293細胞後，易觀察到腫瘤生成現象；其中核心蛋白胺基酸F115-116序列及醣基鏈對於endocan促進腫瘤生長的功能而言十分重要。臨床研究也發現，在許多癌症及敗血休克病人的血液中皆可偵測到高表現量的endocan；其表現量與癌症病人之癒後恢復不良及血管新生現象息息相關，故推測endocan可做為腫瘤嚴重程度的指標。以上研究顯示，endocan可能影響腫瘤生長及伴隨而來的血管新生現象。
為了進一步確定endocan與細胞癌化的關聯性，本研究首先建立穩定表現endocan的RHEK-1細胞株，利用相關in vitro 實驗探討其生長特性、細胞爬行能力及致癌潛能。結果顯示，穩定表現endocan的RHEK-1細胞株不論是生長速率、細胞爬行能力或細胞聚落形成能力，相較母株細胞或載體表現細胞株皆無明顯差異，此一結果顯示endocan似乎無法直接影響細胞癌化路徑。
然而在血管新生的研究上，以含少量血清之endocan條件化上清液培養人類微血管內皮細胞株HMEC-1，偵測內皮細胞增生情形；結果顯示，endocan與少量血清共同作用時可促進內皮細胞生長。此外，含大量endocan之無血清條件化上清液可有效吸引HMEC-1與人類臍帶靜脈內皮細胞HUVEC轉移；於條件化上清液加入endocan抗體後即降低轉移之HMEC-1數目，證明endocan為促進內皮細胞轉移之重要因子。利用endocan突變細胞株進行實驗，結果顯示endocan組成之醣基鏈與核心蛋白胺基酸F115-116序列為其吸引內皮細胞轉移之重要區域。後續實驗發現endocan亦可促進內皮細胞侵犯能力。綜合上述實驗結果，推測endocan參與在血管新生之特定步驟，然而其與腫瘤癌化及血管新生的關聯性仍需進一步於相關實驗中証實。

關鍵字

蛋白多醣 ； 內皮細胞 ； 轉移 ； 血管新生

並列摘要

Endocan （previously called endothelial cell-specific molecule 1, Esm-1） is a 50 kDa soluble proteoglycan that is constituted of a core protein and a single condroitin/dermatan sulfate （CS/DS）chain linked to the serine residue. Endocan is predominantly expressed and secreted by vascular endothelium in response to pro-inflammatory cytokines and pro-angiogenic factors such as TNF-α and VEGF-A. Endocan can bind to the hepatocyte growth factor/scatter factor （HGF/SF） through its glycan domain, thereby enhance HGF/SF mitogenic ability. In mouse model of human tumor xenograft, endocan expression can lead to an increased tumor incidence and this activity of endocan is dependent on its glycan chain moiety and F115-116 residues. Clinical studies indicated that levels of circulating endocan were elevated in several types of cancers and inflammatory disorders, and its expression was correlated with poor prognosis and severity of angiogenic status in cancer patients. In this regard, endocan can be used as a marker of tumor aggressiveness. Previous studies suggest that endocan may be involved in controlling tumor development and play a potential role in angiogenesis accompanied by tumor progression.
Since the exact role of endocan in tumorigenesis is still vague, in the study, we first established endocan-overexpressing RHEK-1 cells lines and studied their growth property, cell migration ability, and tumorigenic potential. Our data indicated that neither growth kinetics nor cell migration ability or transforming ability of these endocan-expressing cell lines were altered when compared to the vector-transfected and parental cell lines. Therefore endocan may not directly involve in the cellular transformation process.
We then tested whether endocan played roles in angiogenesis. Low-serum-containing conditioned medium from endocan-expressing cells or vector-transfected cells were added to the human microvascular endothelial cells （HMEC-1）and the proliferation of HMEC-1 was measured. Our data indicated that endocan could stimulate the growth of HMEC-1 cells in the presence of low serum. Serum-free conditioned medium from endocan-expressing cells were found to be able to stimulate the migration of HMEC-1 and human umbilical vein endothelial cells (HUVEC) in the transwell migration assays. This ability of endocan-containing conditioned medium was indeed attributed to endocan because anti-endocan antibody could block the ability of conditioned medium to promote HMEC-1 migration. Mutant studies indicated that endocan core protein region surrounding F115-116 residues and its GAG chain moiety were important for endocan’s migration-stimulating function. In addition to stimulating the migration of endothelial cells, endocan was also found to be able to stimulate the invasion ability of HMEC-1. Together, these data indicate that endocan does play a role in angiogenesis. Its effect on tumor progression and angiogenesis requires further investigation.

並列關鍵字

proteoglycan ； endothelial cell ； migration ； angiogenesis

參考文獻

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