CAJ | 학술논문

目的观察转化生长因子-β1(TGF-β1)对体外培养的毛囊干细胞(HFSCs)生物学特性的影响.方法体外分离培养人HFSCs,观察其在10-μg/L TGF-β1条件培养基下的细胞形态变化,噻唑蓝(MTT)比色法于24、48、72 h分别检测加入条件培养基前后细胞增殖能力,细胞接近铺满时划痕,并于0、24、48、72 h后用目镜测微尺测量划痕宽度检测迁移能力,将细胞接种于Transwell小室上层,下层加入含10 μg/L TGF-β1条件培养基,48 h后计数穿透细胞数,免疫荧光化学方法检测加入条件培养基前后角蛋白19、β-整合素、E钙黏蛋白、N钙黏蛋白、波形蛋白的表达.结果在TGF-β1作用下,HFSCs之间的紧密连接消失,细胞由圆形或多角形向梭形细胞转变,MTT结果显示,在TGF-β1作用下HFSCs各时间点A值与对照组比较,差异无统计学意义(P>0.05),细胞划痕实验结果显示,TGF-β1能够促进细胞迁移,对照组差异有统计学意义(P<0.01).Transwell穿透实验结果表明,加入TGF-β1后,HFSCs细胞定向迁移穿透基底膜数量与对照组比较显著增多(47.9±8.8比35.8±6.7,P<0.01),免疫荧光结果显示TGF-β1能够降低角蛋白19、β-整合素和E钙黏蛋白表达,而N钙黏蛋白、波形蛋白的表达增加.结论 TGF-β1通过诱导体外培养的HFSCs上皮间质转化而促进其迁移.
목적 관찰전화생장인자-β1(TGF-β1)대체외배양적모낭간세포(HFSCs)생물학특성적영향.방법 체외분리배양인HFSCs,관찰기재10-μg/L TGF-β1조건배양기하적세포형태변화,새서람(MTT)비색법우24、48、72 h분별검측가입조건배양기전후세포증식능력,세포접근포만시화흔,병우0、24、48、72 h후용목경측미척측량화흔관도검측천이능력,장세포접충우Transwell소실상층,하층가입함10 μg/L TGF-β1조건배양기,48 h후계수천투세포수,면역형광화학방법검측가입조건배양기전후각단백19、β-정합소、E개점단백、N개점단백、파형단백적표체.결과재TGF-β1작용하,HFSCs지간적긴밀련접소실,세포유원형혹다각형향사형세포전변,MTT결과현시,재TGF-β1작용하HFSCs각시간점A치여대조조비교,차이무통계학의의(P>0.05),세포화흔실험결과현시,TGF-β1능구촉진세포천이,대조조차이유통계학의의(P<0.01).Transwell천투실험결과표명,가입TGF-β1후,HFSCs세포정향천이천투기저막수량여대조조비교현저증다(47.9±8.8비35.8±6.7,P<0.01),면역형광결과현시TGF-β1능구강저각단백19、β-정합소화E개점단백표체,이N개점단백、파형단백적표체증가.결론 TGF-β1통과유도체외배양적HFSCs상피간질전화이촉진기천이.
Objective To investigate the effects of transforming growth factor (TGF)-β1 on biological activities of hair follicle stem cells (HFSCs) in vitro.Methods The morphological and biological characters of HFSCs were observed when cultured with keratinocyte-serum free medium (K-SFM) containing 10μg/L TGF-β1 and K-SFM in vitro.The proliferative abilities were examined by MTT assay at the time points of 24,48,96 h after the HFSCs were cultured with both medium.The migration ability was measured by wound healing assay at the time points of 0,24,48,72 h.The penetrating cells were calculated at the 48th h after cultured in Transwell with both medium,and the expression of K19,β-intergrin,E-Cadhenin,N-Cadhenin and Vimentin were also detected by immunofluorescence.Results The HFSCs cultured with TGF-β1 lost their typical cobblestone patterns of epithelial cells,displaying spindle-shape,fibroblast-like morphology.The absorbance (A) of HFSCs in each time point had no significant difference between two groups (P＞0.05).The wound healing assay showed that the migration ratio of HFSCs induced by TGF-β1 was higher than in control group (P ＜0.01 ),and the counts of HFSCs penetrating through membrane of Transwell in TGF-β1 group were 47.9 ± 8.8,significantly more than those in the control group [( 35.8 ± 6.7 ),P＜0.01].Immunofluoresence revealed that the expression of K19,β-intergrin and E-Cadhenin was decreased and that of N-Cadhenin and Vimentin was increased in cells stimulated by TGF-β1.Conclusion TGF-β1 can prompt HFSCs migration activity by driving their epithelial-mesenchymal-transition in vitro.