利用基因剔除小鼠探討Hepsin的生理功能

Investigation of the physiological functions of Hepsin using gene knockout mice

徐玉真(Yu-Chen Hsu)

指導教授 ： 林淑華

國立臺灣大學/醫學院/醫學檢驗暨生物技術學研究所/博士(2010年)

https://doi.org/10.6342/NTU.2010.10621

摘要

Hepsin為第二型穿膜絲胺酸蛋白酶，以肝臟細胞的表現量最多。相關文獻指出，Hepsin可能參與肝細胞發育、受精卵著床、血液凝固機轉等過程，但分子機制尚不明確。此外Hepsin在多種癌細胞株中大量表現，尤以前列腺癌最為明顯，除可成為癌細胞的生物標記外，許多證據顯示Hepsin 可能參與癌細胞的生長與轉移，但調控方式仍不清楚。至於hepsin基因剔除小鼠因無明顯生理異常，故Hepsin生理功能尚待研究。
為探討Hepsin蛋白的生理功能，實驗室先前研究發現，進行脾臟內植入黑色素瘤細胞的癌轉移實驗，hepsin基因剔除(KO)小鼠腫瘤細胞轉移至肝臟的聚落數明顯多於野生型(WT)小鼠，且存活率亦降低。我們根據腫瘤細胞發生轉移的過程與影響腫瘤細胞聚落形成的相關原因設計實驗，逐步排除了hepsin基因剔除小鼠可能的異常。同時，經由不同時間點的觀察，發現自脾臟內植入的黑色素瘤細胞或不同直徑的綠色螢光微球體均易留滯於hepsin基因剔除小鼠肝組織。經過穿透式電子顯微鏡及多光子活體內顯微鏡(multiphoton intravital microscope)觀察後，推測易留滯情形是由於hepsin基因剔除小鼠的肝細胞體積變大，造成血管竇直徑較野生型窄所造成。
根據目前實驗結果推測，hepsin基因剔除小鼠肝細胞體積較大，可能是由於體內缺乏Hepsin蛋白活化pro-HGF為肝細胞生長因子，使肝細胞表面間隙接合分子(gap junction)的組成蛋白Connexin 26 (Cx26)、Cx32代謝減緩後，異常堆積所造成。實驗除證明(1)hepsin基因剔除小鼠血清及肝組織中的肝細胞生長因子濃度及肝細胞生長因子受體c-met磷酸化程度均較野生型小鼠低外，也發現(2)hepsin基因剔除小鼠肝組織的Cx26、Cx32蛋白表現量及傳遞功能均較野生型好，(3) Cx26、Cx32蛋白過量表現的肝細胞株體積會變大，及(4)投予hepsin基因剔除小鼠肝細胞生長因子或投予野生型小鼠肝細胞生長因子拮抗劑NK4均能動態調節所觀察之現象。
根據肝細胞生長因子在橫膈膜發育過程十分重要，而hepsin基因剔除小鼠體內的肝細胞生長因子又濃度偏低，實驗也觀察到hepsin基因剔除小鼠橫膈膜發育異常，且導致肝臟往胸腔擠壓，影響肺功能。另外，由於肝細胞表面Cx26、Cx32蛋白表現增加，hepsin基因剔除小鼠在分離初代肝細胞時(EGTA/collagenase perfusion procedure)，細胞存活率及收得活細胞數均較野生型低的現象，可經抑制肝細胞過量表現的間隙接合分子功能，有效提升小鼠初代肝細胞分離後的存活率。
總結實驗結果具有以下重要性：(1)了解肝臟內源性Hepsin蛋白的生理功能與維持肝臟結構或體內肝細胞生長因子濃度的重要性，(2)提供生物體內Hepsin蛋白可活化pro-HGF為肝細胞生長因子的證據，(3)利用活體小鼠，直接提供器官微血管過密或肝臟血管竇過窄，會增加體循環血流中的腫瘤細胞留滯於組織器官的直接模型，且該現象能被動態調節，(4)過去文獻多偏重於Hepsin蛋白與促進癌細胞生長與轉移的關係，甚至多在探討以Hepsin抗體抑制癌轉移的可能應用，而忽略了Hepsin蛋白的生理功能；實驗證明，投予Hepsin抗體可能反而會促進腫瘤細胞轉移至肝臟的風險，需進一步釐清Hepsin蛋白各作用功能區及選擇更適合的抗體以供治療應用。

關鍵字

穿膜絲胺酸蛋白酶腫瘤細胞轉移 ； 間隙接合分子 ； 肝細胞生長因子 ； 老鼠模型

並列摘要

Hepsin, a type II transmembrane serine protease, was indicated involve in hepatocyte growth, blastocyst hatching and blood coagulation pathway. Since there is no obvious abnormality in hepsin-deficient mice, the physiological functions of Hepsin are still unknown. It has also been reported that the overexpression of Hepsin in many cancer cell lines, especially in prostate cancer. Accordindingly, beside be concerned as a biomarker in cancer, many evidences show that Hepsin may play roles in tumor proliferation and metastasis, but the mechanism is not clear so far.
To evaluate the physiological functions of Hepsin, we challenged the tumor metastasis experiment in the hepsin knockout (KO) mice and found that the isogenic tumor cells colonized more efficiently to livers in the KO mice than that in the WT mice.To investigate this mechanism, we used intrasplenic injection and time course tracing strategies showed that both melanoma cells and the different sizes of fluorescent microbeads were more preferentially trapped in hepsin KO livers. With observation through transmission electron microscope and multiphoton intravital microscope, our results showed that the preferential retention of the tumor cells in the KO mice liver is due to the altered hepatic sinusoidal architecture tortured by the enlarged hepatocytes.
To delineate the mechanism responsible for the change of hepacyte sizes, we found the hepsin KO liver had increased Connexin proteins and the increased gap junctions are associated with changes of hepatocyte size. Further studies shown that hepatocyte growth factor (HGF) was associated with the expression level of Connexin proteins and the KO mice had decreased HGF in the liver microcirculation as well as in liver extracts. These observations demonstrate that hepsin involves in liver metastasis by regulating the diameter of the sinusoids and the volume of hepatocytes through its influence on HGF.
Our study provides evidence showing for the first time the relationships between hepsin and HGF/SF in vivo. We also provide a direct evidence of the relationship between successful tumor metastasis and the architecture of target organs. Our findings suggest the application of hepsin protein can be potential therapeutic approach to remission of the liver metastasis.

並列關鍵字

serine protease ； metastasis ； gap junction ； hepatocyte growth factor ； mouse model

參考文獻

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