Sasahara Yusuke, Yoshikawa Yoshie, Morinaga Tomonori, Nakano Yoshiro, Kanazawa Nozomi, Kotani Joji, Kawamata Shin, Murakami Yoshinobu, Takeuchi Katsuyuki, Inoue Chizuko, Kitano Yukio, Hashimoto-Tamaoki Tomoko
Department of Genetics, Hyogo College of Medicine, Nishinomiya, Hyogo 663-8501, Japan.
Int J Oncol. 2009 May;34(5):1191-9.
Human keratinocyte strains derived from the bulge region of plucked human follicles were successfully established from all 43 donors (age 24-76) regardless of the age and gender. The total cell number, number of population doublings and population doubling time were similar among the strains. These bulge-derived keratinocytes, BDKs, expressed keratin family genes specific to basal cell layers of the epidermis. They also expressed CD34, one of the bulge stem cell marker genes. The growth behavior and positivity of CD34 indicate that BDKs contain stem cells. BDKs were cultured until confluency or treated with CaCl2 to induce differentiation. Morphology and expression of keratin family genes in BDKs before and after differentiation induction with CaCl2 were similar to those of epidermal keratinocytes obtained from skin biopsies (NHEKs). However, expression levels of keratin-10, a prickle cell layer marker, in CaCl2-treated BDKs were lower than those in CaCl2-treated NHEKs. Higher expression of integrin-alpha6, a basal cell layer marker, was also noted in BDKs than in NHEKs after differentiation induction. Expression of stem cell marker genes other than CD34, including CD200, Sox2 and NANOG, was about the same at confluency in both cells, but significantly higher in BDKs than NHEKs after differentiation. These results indicate that BDKs were more refractory to differentiation than NHEKs. We then examined Wnt signaling inhibitor genes, DKK-3 and WIF-1 that function as tumor suppressors. DKK-3 expression decreased in both BDKs and NHEKs after CaCl2-induced differentiation. Expression of WIF-1 decreased 50% in BDKs one day after CaCl2 treatment and remained low, but was induced 1.7 times in NHEKs one day after CaCl2 treatment and further induced thereafter (>2.5 times), suggesting that WIF-1 may be involved in maintaining the differentiation-refractory status of BDKs. Since cancer stem cells in the skin have been reported to be similar to bulge-derived stem cells, our BDK strains may be of use in studying characteristics of cancer stem cells of the epidermis.
从43名捐赠者（年龄24 - 76岁）拔取的人毛囊隆突区成功建立了人角质形成细胞系，且不受年龄和性别的影响。各细胞系的总细胞数、群体倍增数和群体倍增时间相似。这些源自隆突区的角质形成细胞（BDK）表达表皮基底层特有的角蛋白家族基因。它们还表达隆突干细胞标记基因之一CD34。CD34的生长行为和阳性表达表明BDK含有干细胞。将BDK培养至汇合或用CaCl₂处理以诱导分化。在用CaCl₂诱导分化前后，BDK中角蛋白家族基因的形态和表达与从皮肤活检获得的表皮角质形成细胞（NHEK）相似。然而，在经CaCl₂处理的BDK中，棘细胞层标记物角蛋白-10的表达水平低于经CaCl₂处理的NHEK。在诱导分化后，BDK中基底细胞层标记物整合素-α6的表达也高于NHEK。除CD34外的干细胞标记基因，包括CD200、Sox2和NANOG，在两种细胞汇合时表达大致相同，但在诱导分化后，BDK中的表达明显高于NHEK。这些结果表明，BDK比NHEK更难分化。然后我们检测了作为肿瘤抑制因子的Wnt信号抑制剂基因DKK - 3和WIF - 1。在CaCl₂诱导分化后，BDK和NHEK中DKK - 3的表达均降低。在CaCl₂处理一天后，BDK中WIF - 1的表达降低50%并维持在低水平，但在CaCl₂处理一天后，NHEK中WIF - 1的表达诱导了1.7倍，此后进一步诱导（>2.5倍），这表明WIF - 1可能参与维持BDK的难分化状态。由于皮肤中的癌症干细胞已被报道与源自隆突区的干细胞相似，我们的BDK细胞系可能有助于研究表皮癌症干细胞的特征。
