探討Kruppel-like Factor 4於犬乳腺腫瘤細胞中的角色

犬乳腺腫瘤(canine mammary gland tumor, cMGT) 是母犬最常發生的腫瘤疾病，而且有高達百分之五十的比例為惡性腫瘤。而且惡性腫瘤的患犬經過手術切除治療後能夠存活超過兩年時間的比例也不及百分之五十。而在現在的醫學技術上，仍然沒有對於惡性的乳腺腫瘤更好的治療方法，所以在本研究中針對可能造成惡性乳腺腫瘤的Kruppel-like factor 4 (KLF4)進行在MGT細胞中的研究，以確定此蛋白質與腫瘤惡性程度相關，以期望以後可以利用此蛋白質進行更深入的研究。在本研究中想要研究的蛋白質KLF4是屬於轉錄因子KLF家族的一員，最早在1996年被發現於腸道跟上皮具有豐富的表現，之後更被發現與細胞的生理功能: 增生、分化、移行或是凋亡有關。而在其中，KLF4在細胞分化的研究上，更被發現可以將已分化成熟的細胞而去分化為具多功能性分化的幹細胞。在本實驗中，我們利用比對大小鼠與人類的已知Klf4序列而設計引子對，而成功地解開844 bp的核酸序列，並且利用這段序列設計專ㄧ的引子對而使用於偵測不同細胞中的Klf4表現，我們發現了MGT細胞跟MDCK細胞都會表現Klf4。另一方面，我們利用了KLF4專ㄧ抗體在正常犬組織切片上進行KLF4的蛋白質標定，發現在犬的腸道跟皮膚上皮也確有較為豐富的表現。之後我們利用Kenpaullone (Ken)進行KLF4的抑制，發現給予Ken八小時後可以成功達到Klf4基因的抑制而在24小時後可以達到蛋白質的抑制。而在KLF4表現量被抑制後，MGT細胞所表現出來的腫瘤惡性程度像是細胞的增生、移行、與群落形成能力相對地下降並且觀察到有細胞週期休止的現象，而這些結果經過計算後也都具有統計上的意義。顯示KLF4對於MGT細胞具有提高腫瘤惡性化的功能。

關鍵字

Kruppel-like factor 4 (KLF4) ；犬乳腺腫瘤； Kenpaullone (Ken)

並列摘要

Canine mammary gland tumor (cMGT) is the most common tumor happened in female dogs and more than 50% of cases were diagnosed as malignant cancers. Though the high mobility and malignancy of MGT are recognized, there are still no effect treatments to elongate the disease-free survival time. Kruppel-like factor 4 (KLF4) is a transcription factor of KLF family and related to cell proliferation, differentiation, migration and apoptosis. Also, KLF4 could induce pluripotent stem cell from differentiated somatic cells with other defined factor. In this study, we identified 844 bp of canine Klf4 sequence from blasting the mRNA of mouse, rat and human and the result was used to designed specific primer pair to detect the Klf4 expression in MGT cells. On the other hand, we detected KLF4 protein expression in normal canine tissue and MGT cells by using polyclonal antibody. We identified KLF4 would abundantly expression in normal canine intestine, skin epithelium and MGT cell lines. In order to realize the role of KLF4 in MGT cells, we downregulated KLF4 expression with Ken and the results suggested Ken would downregulated KLF4 expression in transcriptional and translational level after 8 to 24 hours incubation. Also, the biofunciton assays were tested in Ken-treated MGT cells and the results shown MGT cells would lose the ability of in vitro tumorigenicity and characters of malignancy.

並列關鍵字

Kruppel-like factor 4 (KLF4) ； canine mammary gland tumor (cMGT) ； Kenpaullone (Ken)

參考文獻

Akaogi, K., Y. Nakajima, et al. (2009). "KLF4 suppresses estrogen-dependent breast cancer growth by inhibiting the transcriptional activity of ERα." Oncogene 28(32): 2894-2902.

Al-Hajj, M., M. S. Wicha, et al. (2003). "Prospective identification of tumorigenic breast cancer cells." Proceedings of the National Academy of Sciences 100(7): 3983.

Alenza, D. P., G. R. Rutteman, et al. (1998). "Relation between Habitual Diet and Canine Mammary Tumors in a Case‐Control Study." Journal of veterinary internal medicine 12(3): 132-139.

Aoi, T., K. Yae, et al. (2008). "Generation of pluripotent stem cells from adult mouse liver and stomach cells." Science's STKE 321(5889): 699.

Bain, J., L. Plater, et al. (2007). "The selectivity of protein kinase inhibitors: a further update." Biochem J 408(Pt 3): 297.

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探討Kruppel-like Factor 4於犬乳腺腫瘤細胞中的角色

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