N6-O'2-dibutyryl cAMP對C6神經膠細胞瘤分化之調控

Abstract

神經膠細胞瘤是一種極為常見的惡性腦瘤。目前的處理方式以手術切除及放射治療為主，然而治療效率並不高，其復發的情形及致死率依然居高不下。利用藥物誘導癌細胞分化可能將其導引為正常細胞狀態，是阻止惡化的一種方法。大鼠C6細胞株是常用的一種神經膠細胞瘤研究模式。C6細胞有極快速增殖的特性，並且擁有寡突膠細胞、星狀膠細胞、以及神經元的分化潛能。本實驗根據癌幹細胞的假說，將C6細胞利用球狀培養環境模擬接近癌幹細胞的情形，同時也用一般貼附培養的C6細胞一起比較，觀察二丁酰環腺嘌呤單核苷酸(dbcAMP)對C6細胞的誘導分化成效。 我們利用相位差顯微鏡觀察形態上的變化，並利用細胞免疫螢光染色、西方墨點轉漬、即時定量聚合酶連鎖反應等方法觀察蛋白質及核醣核酸的表現。經過dbcAMP處理，原本呈現多角型的細胞變得狹長，而伸出細長的突起，接近前趨膠細胞的形態。部份在球狀培養的細胞甚至呈現星狀，類似星狀膠細胞或神經元。在蛋白質及核醣核酸的表現上發現，代表星狀膠細胞的標記GFAP在dbcAMP處理後增加；有趣的發現是代表神經元的標識ßⅢ-tubulin在球狀培養的細胞增加表現；同時，代表神經幹細胞的nestin表現則相對減弱。 透過dbcAMP引發C6細胞分化的研究，我們認為實驗所使用的球狀培養細胞方式，能促使細胞擁有癌幹細胞的特質。這樣的模式將可運用在C6癌幹細胞起始狀態及分化路徑的研究。

Glioma is one of the most common primary brain tumors. Its conventional treatments such as surgical resection or radiation fail to increase the survival of patients. Induction of differentiation is one way for controlling the malignant growth of glioma. Rat C6 glioma is a rapidly proliferating cell line with oligodendrocytic, astrocytic, and neuronal properties. Based on the cancer stem cell hypothesis, in this study, we use C6 tumor sphere cells, together with parental adhesion cells, to investigate the regulation of differentiation-inducing agent, N6-O'2-dibutyryl cAMP (dbcAMP). The efficacy of treatment was examined by phase-contrast microscopy, Immunocytochemistry, Western blot, RT-PCR and Real Time Quantitative PCR. After dbcAMP treatment, morphological changes of C6 cells could be easily observed. Most of the adhesion cells showed bipolar shape, which are similar to glial progenitors. Compared with adhesion cells, sphere cells exposed to dbcAMP also extended processes, and some cells resulted in a stellar shape. The morphology more closely resembled mature astrocyte or neuron. Astrocytic marker glial fibrillary acidic protein (GFAP) expression increased in tumorsphere. Interestingly, the specific neuronal marker ßⅢ-tubulin immunopositive cells were also observed. Meanwhile, the neural stem cell marker nestin decreased relatively. Our results suggest that the formation of tumor sphere model, which has cancer stem-like cells potential, could be a suitable approach to identify the initiation and progression of rat C6 glioma cells.