應用脂肪幹細胞的腫瘤趨向性攜帶反轉錄病毒載體以治療腦腫瘤

膠質瘤是最常見的原發性腦腫瘤，世界衛生組織將膠質瘤依惡性程度分為四個等級，其中以第四級的膠質母細胞瘤（Glioblastoma）最為惡性、患者預後最差。因為GBM有浸潤性，癌細胞會入侵正常的腦組織，導致手術治療無法完全將癌細胞清除，使患者復發率非常的高，即使接受了治療，預後大約也只有一年。最近研究指出，幹細胞很有希望能夠成為腦瘤治療的載體。實驗室過去已經證實，人類脂肪幹細胞（hASC）能夠運送病毒載體到腫瘤位置，並且釋放出反轉錄病毒（RRV）感染腫瘤。為了進一步驗證這個結果，我會進行裸鼠顱內 hASC 往腦腫瘤遷移的實驗，分別以綠螢光蛋白（GFP）與紅螢光蛋白（dsRed）標記 hASC 與人類腦癌細胞（U87），確認 hASC 在顱內會受到腫瘤的吸引而移動。實驗室過去也做了［3-（4,5-dimethylthiazol-2-yl）-5-（3-carboxymethoxyphenyl）-2-（4-sulfophenyl）-2H-tetrazolium,innersalt， MTS］ assay 確定人類腦癌細胞（U87）被 hASC 釋放的 ACE-CD 病毒感染，搭配 5-FC 毒殺的效率。所以接著我會在裸鼠顱內建立 U87 腦瘤，之後再利用 hASC-ACE-CD 搭配 5-FC 進行原位腫瘤的治療實驗，可以看到開始注射 5-FC 進行治療後，老鼠的生存時間明顯延長，治療組老鼠的存活中位數為 128.5 天比控制組老鼠存活中位數的 50 天多了一倍以上，P < 0.001 。為了能夠在實驗中更直接地觀察顱內腫瘤大小的變化，我也建構了帶有遠紅外光蛋白（iRFP）的慢病毒載體，並且感染 U87 ，使 U87 帶有遠紅外光蛋白，再利用螢光分子斷層成像 ( Fluorescence Molecular Tomography ) 進行活體造影，觀察 U87 在老鼠顱內的生長，並且追蹤治療的效果。透過 FMT 造影，可以看到隨著老鼠顱內植入腫瘤的時間越長， iRFP 的訊號就越強，而在追蹤治療效果的過程中，可以看到老鼠施打 5-FC 後，腫瘤的訊號會被抑制，並且隨著治療的進行能進一步使腫瘤訊號縮小、甚至消失，表示治療能使腫瘤縮小並毒殺腫瘤。我們希望利用hASC攜帶RRV的方式，做為改善目前GBM治療的方案。

關鍵字

基因治療；脂肪幹細胞；腦瘤；活體影像斷層掃描

並列摘要

Glioblastoma multiforme (GBM) has universally poor prognosis that is thought to be related to cellular invasion into normal tissue, making localized treatments like surgery and radiation insufficient to treat the total burden of disease. Recently, stem cells have been found offering much promise as delivery vehicles for brain tumor therapy. Our previous study had confirmed the tumor-tropic migratory ability of human adipose-derived stem cells (hASC) and investigated the efficiency of hASC to deliver replicating retrovirus vector (RRV) into glioma cells in vitro and in vivo. This in vivo experiment will be repeated for validation. Our previous study also had determined the therapeutic effects of hASC-ACE-CD (RRV expressing the yeast cytosine deaminase suicide gene) by MTS assay and an orthotopic mouse model of human glioma after treatment with prodrug 5-FC. The therapeutic effect can also delay the onset of animals. To further validate this result, we will repeat this in vivo experiment, too. In order to more intuitively observe the changes in tumor size, we constructed a lentiviral vector containing near infrared fluorescent protein (iRFP) and transduced the viral vector into U87 cells followed by subcutaneous inoculation of the cells in nude mice. Then, we used the Fluorescence Molecular Tomography (FMT) to detect the iRFP signals for observation of the tumor growth and to track the effectiveness of treatment. We wish the use of hASC as the vehicle for dispersing RRV may provide a viable strategy for glioma therapy.

並列關鍵字

Gene therapy ； Adipose derived stem cells ； Glioma ； Fluorescent molecular tomography

參考文獻

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應用脂肪幹細胞的腫瘤趨向性攜帶反轉錄病毒載體以治療腦腫瘤

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