大腸癌幹細胞新穎標記之研究

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2017

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摘要 大腸直腸癌是世界上最常見的癌症之一。雖現已研發出一些新治療方式及策略,但對大多數中晚期階段的患者而言,預後狀況仍不樂觀。近年來的研究發現有一小群具有類似幹細胞特性的腫瘤細胞,稱為癌幹細胞,與腫瘤的生成、復發、轉移及對化學放射治療產生抗性有主要關係。因此,能夠找出具專一辨識性的大腸癌幹細胞生物標記是非常切要的。CD133是目前相當普遍被用來辨識和分離癌幹細胞的一種生物標記,但對於其能否作為大腸癌幹細胞的辨識標記仍存有許多爭議,所以我們想要找出其它更專一的大腸癌幹細胞生物標記。目前我們收集了102個中晚期階段不同病人手術切下的腫瘤檢體進行初級細胞培養,包括培養出腫瘤球 (tumorspheres) 與貼盤細胞然後與原來的組織塊一起做mRNA分析,希望能藉此找出與大腸癌幹細胞密切相關的因子。我們篩選出最能以癌幹細胞球繼代培養的2811細胞株,抽取其懸浮培養的腫瘤球及貼盤細胞之RNA,和2901的新鮮腫瘤組織塊之RNA去進行 microarray分析比較。發現驅動蛋白12 (Kinesin Family Member 12, KIF12) 基因的mRNA表現量在懸浮培養的腫瘤球內比起貼盤培養的細胞與腫瘤組織塊要高。我們接著在其他臨床檢體的初級培養細胞中檢測KIF12的mRNA及蛋白質的表現量,結果顯示KIF12的mRNA含量在4株我們初級培養出的人類大腸癌類癌幹細胞中的表現量確實比較高。另外,利用人類大腸癌細胞株HCT-116及HT-29衍生出之類癌幹細胞球亦有同樣的結果。然而,利用Western blot分析其蛋白質表現的結果顯示,KIF12蛋白質在HT-29的類癌幹細胞球中比起貼附的癌細胞確實有明顯升高,但在HCT-116中卻相反。因HT-29與HCT-116細胞株主要差別為HT-29為p53突變之細胞,因此KIF12之表現量與p53之關聯尚須更多研究。根據以上結果,我們希望探討利用KIF12作為大腸癌幹細胞的生物標記之可能性,期望未來能夠提供臨床醫療應用。
Abstract Colorectal cancer (CRC) is one of the most common cancers in the world. Despite there are different treatments developed, a high percentage of patients with advanced tumor still have poor prognosis. Recent studies have found that a small population of tumor cells, known as cancer stem cells (CSCs), may be responsible for the tumorigenesis, recurrence, metastasis and resistance of chemoradiotherapy. Therefore, it is critical to find the biomarkers of CRC stem cells (CRCSCs). CD133 is currently a widely-used marker to identify and isolate CSCs. However, the use of CD133 in the identification of CRCSCs is still controversial. As a result, we prospect to identify more specific and reliable biomarkers for CRCSCs. We collected 102 fresh surgical specimens from patients with the middle to late stages of CRC, and sub-cultured them to tumorspheres. Meanwhile, we also established the adherent primary cancer cell culture. Then, we analyzed the mRNA of them, and expected to find some key factors which are highly correlated with CRCSCs. The tumorspheroid cell line 2811 with the strongest proliferation ability of all the tested samples was selected and subjected to mRNA microarray analysis. Comparing with its adherent primary cancer cells and 2901unprocessed tumor tissue sample, the kinesin family member 12 (KIF12) had much more expression in the tumorspheres than in the adherent cells and the tumor tissue. Next, we also tested the expressions of the protein and mRNA levels of KIF12 in the four clinical CRC primary cells derived from the other patients. The results suggested that the mRNA levels of KIF12 were really higher in the tumorspheres we cultured. In addition, similar results were observed in two human CRC cell lines, HT-29 and HCT-116. However, the expressions of KIF12 protein were only accumulated in the tumorspheres derived from HT-29, but oppositely decreased in the tumorspheres of HCT-116. The main difference between HT-29 and HCT-116 is that HT-29 has mutant p53. The correlation of KIF12 and p53 needs more studies to elucidate. In summary, KIF12 has the potential for being developed as one of the biomarkers for CRCSCs for clinical applications in the future.

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大腸直腸癌, 大腸直腸癌幹細胞, 生物標記, 驅動蛋白12, colorectal cancer (CRC), colorectal cancer stem cells (CRCSCs), biomarkers, kinesin family member 12 (KIF12)

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