CAJ | 학술논문

背景：毛囊干细胞的增殖分化受到自身基因及外来信号的共同作用，Wnt/β-catenin信号通路在毛囊毛发发育中起重要作用，但详细机制尚未明确。 　　目的：探讨在角质细胞生长因子及氯化锂干预下，Wnt/β-catenin信号通路在人毛囊干细胞向毛囊乳突细胞或表皮细胞定向分化中的作用及与其他信号分子的相互关系。 　　方法：获取毛囊隆突区干细胞进行培养，检测其生长曲线，观察1×106 L-1、1×107 L-1、1×108 L-1和1×109 L-1培养的毛囊干细胞在不同时间点的增殖效应；使用免疫荧光染色法对毛囊干细胞及其分化细胞进行鉴定。分别以0，0.5，1.5，10，25 mmol/L氯化锂及0，10，25，50，100μg/L角质细胞生长因子诱导毛囊干细胞分化，对比各组细胞的增殖效应，探索促毛囊干细胞分化的最佳氯化锂及角质细胞生长因子浓度。使用RT-PCR检测未处理对照组、10 mmol/L氯化锂组和10μg/L角质细胞生长因子组干预后3，5，7，9 d细胞的β-catenin、APC、GSK-3β、Axin和Lef1的mRNA转录水平。 　　结果与结论：分离培养的毛囊干细胞在体外经多次传代后仍具有很强的增殖能力和多向分化潜能，随氯化锂浓度升高，细胞增殖效应减弱；而随角质细胞生长因子组质量浓度增高细胞增殖能力增强。含有氯化锂的K-SFM条件培养基中毛囊干细胞形态改变明显，各组间有明显差别，氯化锂>10 mmol/L时分化比例高，β-catenin表达量增高；而含有角质细胞生长因子K-SFM条件培养基中毛囊干细胞向表皮细胞分化，β-catenin变化不明显。提示氯化锂在促毛囊干细胞分化中，激活 Wnt/β-catenin 信号通路，抑制降解复合物重要成分GSK-3β的表达下降，促使β-catenin在细胞浆表达增加并转入核内，增加靶基因转录，促使毛囊干细胞向毛囊细胞方向分化。氯化锂>10 mmol/L是促毛囊干细胞分化的最佳浓度，但增殖效应减弱。角质细胞生长因子可促进毛囊干细胞向表皮分化，可促进毛囊干细胞增殖和迁移，促进创面再上皮化及创面愈合。氯化锂和角质细胞生长因子促毛囊干细胞定向分化的作用机制可能为激活Wnt/β-catenin信号通路，促使β-catenin表达改变，从而激活Wnt信号通路中Lef介导相关靶基因的转录。揹景：毛囊榦細胞的增殖分化受到自身基因及外來信號的共同作用，Wnt/β-catenin信號通路在毛囊毛髮髮育中起重要作用，但詳細機製尚未明確。 　　目的：探討在角質細胞生長因子及氯化鋰榦預下，Wnt/β-catenin信號通路在人毛囊榦細胞嚮毛囊乳突細胞或錶皮細胞定嚮分化中的作用及與其他信號分子的相互關繫。 　　方法：穫取毛囊隆突區榦細胞進行培養，檢測其生長麯線，觀察1×106 L-1、1×107 L-1、1×108 L-1和1×109 L-1培養的毛囊榦細胞在不同時間點的增殖效應；使用免疫熒光染色法對毛囊榦細胞及其分化細胞進行鑒定。分彆以0，0.5，1.5，10，25 mmol/L氯化鋰及0，10，25，50，100μg/L角質細胞生長因子誘導毛囊榦細胞分化，對比各組細胞的增殖效應，探索促毛囊榦細胞分化的最佳氯化鋰及角質細胞生長因子濃度。使用RT-PCR檢測未處理對照組、10 mmol/L氯化鋰組和10μg/L角質細胞生長因子組榦預後3，5，7，9 d細胞的β-catenin、APC、GSK-3β、Axin和Lef1的mRNA轉錄水平。 　　結果與結論：分離培養的毛囊榦細胞在體外經多次傳代後仍具有很彊的增殖能力和多嚮分化潛能，隨氯化鋰濃度升高，細胞增殖效應減弱；而隨角質細胞生長因子組質量濃度增高細胞增殖能力增彊。含有氯化鋰的K-SFM條件培養基中毛囊榦細胞形態改變明顯，各組間有明顯差彆，氯化鋰>10 mmol/L時分化比例高，β-catenin錶達量增高；而含有角質細胞生長因子K-SFM條件培養基中毛囊榦細胞嚮錶皮細胞分化，β-catenin變化不明顯。提示氯化鋰在促毛囊榦細胞分化中，激活 Wnt/β-catenin 信號通路，抑製降解複閤物重要成分GSK-3β的錶達下降，促使β-catenin在細胞漿錶達增加併轉入覈內，增加靶基因轉錄，促使毛囊榦細胞嚮毛囊細胞方嚮分化。氯化鋰>10 mmol/L是促毛囊榦細胞分化的最佳濃度，但增殖效應減弱。角質細胞生長因子可促進毛囊榦細胞嚮錶皮分化，可促進毛囊榦細胞增殖和遷移，促進創麵再上皮化及創麵愈閤。氯化鋰和角質細胞生長因子促毛囊榦細胞定嚮分化的作用機製可能為激活Wnt/β-catenin信號通路，促使β-catenin錶達改變，從而激活Wnt信號通路中Lef介導相關靶基因的轉錄。
배경：모낭간세포적증식분화수도자신기인급외래신호적공동작용，Wnt/β-catenin신호통로재모낭모발발육중기중요작용，단상세궤제상미명학。 　　목적：탐토재각질세포생장인자급록화리간예하，Wnt/β-catenin신호통로재인모낭간세포향모낭유돌세포혹표피세포정향분화중적작용급여기타신호분자적상호관계。 　　방법：획취모낭륭돌구간세포진행배양，검측기생장곡선，관찰1×106 L-1、1×107 L-1、1×108 L-1화1×109 L-1배양적모낭간세포재불동시간점적증식효응；사용면역형광염색법대모낭간세포급기분화세포진행감정。분별이0，0.5，1.5，10，25 mmol/L록화리급0，10，25，50，100μg/L각질세포생장인자유도모낭간세포분화，대비각조세포적증식효응，탐색촉모낭간세포분화적최가록화리급각질세포생장인자농도。사용RT-PCR검측미처리대조조、10 mmol/L록화리조화10μg/L각질세포생장인자조간예후3，5，7，9 d세포적β-catenin、APC、GSK-3β、Axin화Lef1적mRNA전록수평。 　　결과여결론：분리배양적모낭간세포재체외경다차전대후잉구유흔강적증식능력화다향분화잠능，수록화리농도승고，세포증식효응감약；이수각질세포생장인자조질량농도증고세포증식능력증강。함유록화리적K-SFM조건배양기중모낭간세포형태개변명현，각조간유명현차별，록화리>10 mmol/L시분화비례고，β-catenin표체량증고；이함유각질세포생장인자K-SFM조건배양기중모낭간세포향표피세포분화，β-catenin변화불명현。제시록화리재촉모낭간세포분화중，격활 Wnt/β-catenin 신호통로，억제강해복합물중요성분GSK-3β적표체하강，촉사β-catenin재세포장표체증가병전입핵내，증가파기인전록，촉사모낭간세포향모낭세포방향분화。록화리>10 mmol/L시촉모낭간세포분화적최가농도，단증식효응감약。각질세포생장인자가촉진모낭간세포향표피분화，가촉진모낭간세포증식화천이，촉진창면재상피화급창면유합。록화리화각질세포생장인자촉모낭간세포정향분화적작용궤제가능위격활Wnt/β-catenin신호통로，촉사β-catenin표체개변，종이격활Wnt신호통로중Lef개도상관파기인적전록。
BACKGROUND:The proliferation and differentiation of hair-folicle-generating stem cels are influenced by the joint action of their own genes and external signals. Wnt/β-catenin signaling pathway plays an important role in the development of hair folicles, but the detailed mechanisms are not yet clear. OBJECTIVE:To investigate, with interruption of keratinocyte growth factor and lithium chloride, the function and the interrelationship of Wnt/β-catenin signaling pathway with other signal factors when human hair-folicle-generating stem cels differentiate into dermal papila cels or epidermal cels. METHODS: Hair-folicle-generating stem cels were isolated from the bulge and cultivated. Then the growth curve of hair-folicle-generating stem cels was tested and formed in order to observe the cellproliferation ability cultivated at different densities (1×106/L, 1×107/L, 1×108/L, 1×109/L) at each time. Immunoflurorescene staining was performed to identify hair-folicle-generating stem cels and their differentiated cels. Lithium chloride (0, 0.5, 1.5, 10, 25 mmol/L individualy) and keratinocyte growth factor(0, 10, 25, 50, 100 μg/L individualy) were used to induce the differentiation of hair-folicle-generating stem cels. Then, we contrasted and analyzed the proliferation ability in each case, thereby investigating the most appropriate concentration of keratinocyte growth factor and lithium chloride to spur the differentiation of hair-folicle-generating stem cels. At days 3, 5, 7 and 9, we tested and compared the mRNA expressions of β-catenin, APC, GSK-3β, Axin and Lef1 from cels in control group, 10 mmol/L lithium chloride group and 10 μg/L keratinocyte growth factor group. RESULTS AND CONCLUSION:Isolating cultured hair-folicle-generating stem cels stil had a great reproductive activity and multi-lineage potential even after various times subculturein vitro. With higher lithium chloride concentration, the proliferation ability of hair-folicle-generating stem cels declined; while it increased when keratinocyte growth factor concentration increased. In K-SFM medium which contained lithium chloride, the transformation of hair-folicle-generating stem cels was obvious, showing distinct differences among groups. Especialy, the level ofβ-catenin reached the peak when lithium chloride > 10 mmol/L. However, in K-SFM medium which contained keratinocyte growth factor, hair-folicle-generating stem cels differentiated into epidermal cels and the level of β-catenin changed slightly. We found that, while spurring the differentiation of hair-folicle-generating stem cels, lithium chloride could activate Wnt/β-catenin signal pathway and inhibit GSK-3β, a vital component of degradation compound. This facilitated β-catenin expressing in the cytoplasm to translocate into the nucleus. As a result, the transcription of target gene increased. It is the most appropriate concentration to spur hair-folicle-generating stem cels differentiation when lithium chloride level is > 10 mmol/L, but the proliferation ability declines correspondingly. Keratinocyte growth factor, which can facilitate hair-folicle-generating stem cels differentiated into epidermal cels, is a key factor to accelerate proliferation ability and migration of hair-folicle-generating stem cels, re-epithelialization and healing of wound. The mechanisms of hair-folicle-generating stem cels oriented differentiation induced by lithium chloride and keratinocyte growth factor are activating Wnt/β-catenin signal pathway, inducing change of β-catenin expression, and activating the transcription of target gene related to Wnt/β-catenin signaling pathway .