探討Leukocyte Cell-Derived Chemotaxin 2 (LECT2) 對於血管內皮生長因子誘發血管新生之影響

Effects of Leukocyte Cell-Derived Chemotaxin 2 (LECT2) on VEGF165-Induced Angiogenesis

鍾宛瑾(Wan-Ching Chung)

指導教授 ： 郭明良

國立臺灣大學/醫學院/毒理學研究所/碩士(2008年)

https://doi.org/10.6342/NTU.2008.02578

摘要

血管新生與癌症發展有密切的關聯，血管新生是一種生理過程，主要作用來自於血管最內層的內皮細胞。當內皮細胞受到血管新生物質的活化而產生細胞增生、移動、萌芽並進一步重組形成新的血管，藉此提供腫瘤生長所需之養分。若能找出抑制血管新生作用之蛋白分子，對於癌症治療將有重大突破。實驗室先前研究利用生物晶片技術(microarray)，找到調控肝癌血管侵犯表現的基因 (leukocyte cell-derived chemotaxin 2，LECT2)。分析127名肝癌病人發現LECT2表現量與腫瘤大小呈現逆相關，進一步以動物實驗與雞胚胎卵黃囊試驗法(chicken chorioallantoic membrane，CAM assay)，發現LECT2具有抑制肝癌腫瘤生成與血管形成之作用。故本實驗欲探討LECT2是否會標的血管內皮細胞，影響血管新生物質誘發血管新生的作用，進一步廣泛性的探討LECT2於多種癌細胞株之血管新生的角色。
本研究以VEGF165為誘發因子，結果顯示LECT2重組蛋白能減少VEGF165所誘發血管內皮生長因子接受器2 (VEGFR2) 磷酸化而抑制下游ERK及AKT的訊息傳遞途徑，使內皮細胞的增生、移動與新生血管的能力下降。此外，CAM assay 與小黑鼠體內血管新生的實驗(Matrigel Plug assay)，同樣觀察到LECT2重組蛋白抑制VEGF165所誘發血管生成。在血管滲透的研究發現，無論在in vitro或in vivo實驗模式皆證明LECT2重組蛋白可以降低VEGF165所誘發的血管滲透作用。收集不同種類癌細胞株的細胞培養液與LECT2重組蛋白處理於內皮細胞，結果顯示LECT2重組蛋白能抑制SK-Hep1肝癌細胞、A549肺癌細胞、MCF-7乳癌細胞以及B16F1小鼠黑色素瘤細胞之血管新生。進一步在B16F1細胞株穩定表現LECT2，結果發現可抑制腫瘤血管增生、降低腫瘤血管密度以達到抑制腫瘤生長的效果。
本研究首先證實LECT2可以直接影響內皮細胞，抑制VEGF165所誘發之血管生成，同時減少腫瘤血管新生之現象。由本研究之結果，更明確闡述LECT2在抗血管新生的角色上所具有的生物效應與作用機轉，此結果對於腫瘤的臨床治療與應用勢必具相當大的潛力與期待，未來進一步將LECT2發展成為蛋白質類藥物，以應用在抗血管新生作用的癌症治療上。

關鍵字

血管新生 ； 內皮細胞 ； 白血球趨化蛋白2

並列摘要

Angiogenesis is a process of new blood vessel formation which has essential roles in development, reproduction and repair. In our previous studies, several that the leukocyte cell-derived chemotaxin 2 (LECT2) gene expression was down-regulated with vascular invasion. The levels of LECT2 are clearly correlated with regulating tumor size and overall survival of HCC patients. Using CAM assay, we found that LECT2-transfected cells conditioned media exhibited extremely low angiogenic activity as compared to control cells. This result suggesting that LECT2 may down-regulate certain angiogenic factors. Hence we proposed to investigate the molecular mechanism underlying LECT2 mediated anti-angiogenic factors-induced angiogenesis in vitro and in vivo. Further, we also observed report the expression of LECT2 in others cancer cells.
In this study we evaluated the function of recombinant human LECT2 (rLECT2) proteins on angiogenesis by using human umbilical vein endothelial cells (HUVEC). We demonstrated a selective and significant inhibition of VEGF165 mediated angiogenic activity in HUVEC by rLECT2 protein through inhibiting the VEGF165-induced proliferation, migration and tube formation. The rLECT2 protein also suppressed VEGF165-induced angiogenesis in CAM assay and matrigel plug assay. Both in vitro and in vivo, we found that LECT2 suppressed the VEGF165-induced vascular permeability. Our results demonstrated that rLECT2 protein could reduce the VEGF165-induced VEGFR-2 phosphorylation and inhibited the expression of downstream ERK and AKT phosphorylation in HUVEC. In addition, rLECT2 protein reduced cancer cell conditioned media-induced tube formation in HUVEC and LECT2 also decreased tumor growth of melanoma cells.
In conclusion, we for the first time found that LECT2 played an important role in anti-angiogenesis. Moreover the LECT2 might have broad therapeutic applications in diseases characterized by excessive angiogenesis.

並列關鍵字

angiogenesis ； endothelial cells ； leukocyte cell-derived chemotaxin 2

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