CAJ | 학술논문

目的观察低频电磁场(LFEMF)对三维环境中培养的人表皮干细胞(ESCs)增殖的影响，为LFEMF用于皮肤组织工程提供实验依据。方法取人包皮来源的ESCs作为种子细胞，利用Ⅳ型胶原快速贴壁法体外分离纯化ESCs，并种植于胶原海绵和壳聚糖支架材料，外加LFEMF治疗仪（场强5 mT，频率分别为1 Hz、10 Hz和50 Hz）刺激ESCs(30 min/d)，用四甲基偶氮唑盐微量酶反应比色法(MTT)检测支架材料细胞毒性，通过肉眼、HE染色后DAPI荧光核染色后镜下观察及扫描电镜观察ESCs生长情况，并收集培养2，7，10,14 d ESCs的DAPI核荧光染色数据，用显微图像软件分析细胞增殖数目，比较各时间点细胞增殖的区别。结果 LFEMF促进ESCs增殖的作用在不同频率组间的差异和两种支架上培养的ESCs增殖情况的差异均有统计学意义(P＜0.01)，10 d后胶原海绵支架上细胞增殖较壳聚糖支架上的细胞明显(P＜0.05)，各频率组LFEMF刺激均能促进ESCs增殖，频率不同，促增殖的效应不一(P ＜0.05)。结论胶原海绵可较好地作为体外培养ESCs的支架，在LFEMF干预下可实现ESCs体外三维环境中较快扩增，LFEMF在皮肤组织工程中具有较大应用潜力。
목적 관찰저빈전자장(LFEMF)대삼유배경중배양적인표피간세포(ESCs)증식적영향，위LFEMF용우피부조직공정제공실험의거。방법 취인포피래원적ESCs작위충자세포，이용Ⅳ형효원쾌속첩벽법체외분리순화ESCs，병충식우효원해면화각취당지가재료，외가LFEMF치료의（장강5 mT，빈솔분별위1 Hz、10 Hz화50 Hz）자격ESCs(30 min/d)，용사갑기우담서염미량매반응비색법(MTT)검측지가재료세포독성，통과육안、HE염색후DAPI형광핵염색후경하관찰급소묘전경관찰ESCs생장정황，병수집배양2，7，10,14 d ESCs적DAPI핵형광염색수거，용현미도상연건분석세포증식수목，비교각시간점세포증식적구별。결과 LFEMF촉진ESCs증식적작용재불동빈솔조간적차이화량충지가상배양적ESCs증식정황적차이균유통계학의의(P＜0.01)，10 d후효원해면지가상세포증식교각취당지가상적세포명현(P＜0.05)，각빈솔조LFEMF자격균능촉진ESCs증식，빈솔불동，촉증식적효응불일(P ＜0.05)。결론 효원해면가교호지작위체외배양ESCs적지가，재LFEMF간예하가실현ESCs체외삼유배경중교쾌확증，LFEMF재피부조직공정중구유교대응용잠력。
Objective To observe the effects of low frequency electromagnetic fields (LFEMFs) on the proliferation of human epidermal stem cells (hESCs) cultured in a three dimensional environment so as to provide an experimental basis for applying LFEMF in skin tissue engineering.Methods hESCs from human prepuces were isolated and purified by the method of rapid adherence to collagen type ⅣV. They were grafted into a type-I collagen sponge or chitosan scaffold in vitro, and then stimulated with different frequencies of LFEMF ( 1 Hz, 10 Hz or 50 Hz) at a magnetic field intensity of 5 mT for 30 min/d. The cells' growth and proliferation were tracked using hematoxylin and eosin (HE) and diamine pheny1 indole (DAPI) staining and observed under the scanning electron microscope at different time points ( on 2nd, 7th, 10th and 14th days of LFEMF intervention). The amounts of cell proliferation at every time point were analyzed and compared.Results LFEMFs of different frequencies showed significantly different efficacy in promoting hESC proliferation. The two scaffolds also showed significantly different effects.By the 10th day, hESCs had grown significantly better on collagen sponge scaffolds than on the chitosan ones. All LFEMF frequencies could promote proliferation of hESCs, but the differences in their effects were statistically significant.Conclusion Collagen sponge may be a preferable scaffold for hESCs cultured in vitro. Rapid proliferation of ESCs in three-dimensional settings can be promoted by LFEMF intervention. LFEMF has relatively great potential in skin tissue engineering.