CAJ | 학술논문

背景：由于人胎盘来源的间充质干细胞具有多方面的优点，近年来已成为干细胞研究的热点。目的：分析鉴定羊膜间充质干细胞和羊膜上皮细胞的生物学特性，探讨其作为皮肤种子细胞在三维气液培养构建组织工程皮肤中的应用情况。方法：用胰酶胶原酶多步消化法获取羊膜间充质干细胞和羊膜上皮细胞，通过流式细胞术、反转录-聚合酶链反应和免疫荧光染色技术，鉴定两种细胞的表面分子标记、干细胞特性、与皮肤角质形成细胞的相似性，并利用两种细胞为种子细胞以鼠Ⅰ型胶原为基质进行三维气液培养。结果与结论：①流式细胞术检测体外培养羊膜间充质干细胞和羊膜上皮细胞均高表达CD90、CD73、CD105，不表达造血干细胞标志CD34以及MHC-Ⅱ类分子HLA-DR。②反转录-聚合酶链反应检测到羊膜间充质干细胞表达干细胞特性基因CMCY和NANOG，羊膜上皮细胞表达干细胞特性基因CMCY和 KLF4，两种细胞均有干细胞特性。③反转录-聚合酶链反应检测羊膜间充质干细胞表达皮肤角质形成细胞特性基因 K19、β1-integrin、K8，羊膜上皮细胞表达K19、β1-integrin、K5、K8，免疫荧光染色见羊膜上皮细胞表达与角质形成细胞增殖相关的的特性蛋白 K14，说明羊膜上皮细胞与皮肤角质形成细胞更具相似性,在特定条件下更易于分化为皮肤角质形成细胞。④利用两种细胞成功构建组织工程皮肤，苏木精-伊红染色切片显示其具有一定的皮肤结构，且羊膜上皮细胞发生了初步分化。以上结果说明羊膜间充质干细胞与羊膜上皮细胞通过三维培养构建人皮肤组织是可行的。
배경：유우인태반래원적간충질간세포구유다방면적우점，근년래이성위간세포연구적열점。목적：분석감정양막간충질간세포화양막상피세포적생물학특성，탐토기작위피부충자세포재삼유기액배양구건조직공정피부중적응용정황。방법：용이매효원매다보소화법획취양막간충질간세포화양막상피세포，통과류식세포술、반전록-취합매련반응화면역형광염색기술，감정량충세포적표면분자표기、간세포특성、여피부각질형성세포적상사성，병이용량충세포위충자세포이서Ⅰ형효원위기질진행삼유기액배양。결과여결론：①류식세포술검측체외배양양막간충질간세포화양막상피세포균고표체CD90、CD73、CD105，불표체조혈간세포표지CD34이급MHC-Ⅱ류분자HLA-DR。②반전록-취합매련반응검측도양막간충질간세포표체간세포특성기인CMCY화NANOG，양막상피세포표체간세포특성기인CMCY화 KLF4，량충세포균유간세포특성。③반전록-취합매련반응검측양막간충질간세포표체피부각질형성세포특성기인 K19、β1-integrin、K8，양막상피세포표체K19、β1-integrin、K5、K8，면역형광염색견양막상피세포표체여각질형성세포증식상관적적특성단백 K14，설명양막상피세포여피부각질형성세포경구상사성,재특정조건하경역우분화위피부각질형성세포。④이용량충세포성공구건조직공정피부，소목정-이홍염색절편현시기구유일정적피부결구，차양막상피세포발생료초보분화。이상결과설명양막간충질간세포여양막상피세포통과삼유배양구건인피부조직시가행적。
BACKGROUND: Placenta mesenchymal stem cells have become hot spots in stem cells study in recent years because of its advantages. OBJECTIVE:To analyze the biological characteristics of amniotic mesenchymal stem cells and amnion epithelial cells, and to explore the feasibility of amniotic mesenchymal stem cells and amnion epithelial cells applied as seed cells in three-dimensional liquid culture to construct the tissue-engineered skin. METHODS:The amniotic mesenchymal stem cells and amnion epithelial cells were obtained by using multi-step digestion with trypsin and col agenase;then the flow cytometry, reverse transcription-PCR and immunofluorescent staining techniques were adopted to identify the surface molecular markers, stem cellcharacteristics and keratinocytes similarity respectively. Based on these data, amniotic mesenchymal stem cells and amnion epithelial cells used as seed cells together with rat type Ⅰ col agen matrix were adopted for three-dimensional liquid culture. RESULTS AND CONCLUSION:Flow cytometry test showed that amniotic mesenchymal stem cells and amnion epithelial cells cultured in vitro could highly express CD90, CD73 and CD105, and could not express the hemopoietic stem cellmarker of CD34 and MHC-class Ⅱ molecular HLA-DR. Reverse transcription-PCR results detected that amniotic mesenchymal stem cells could express stem cellcharacteristic genes CMCY and NANOG;amnion epithelial cells could express stem cellcharacteristic genes CMCY and KLF4, showing that both amniotic mesenchymal stem cells and amnion epithelial cells have stem cellproperties. Reverse transcription-PCR results showed that amniotic mesenchymal stem cells could express keratinocytes characteristic genes K19,β1-integrin and K8;amnion epithelial cells could express K19,β1-integrin, K5 and K8. Immunofluorescence staining results showed amnion epithelial cells could express keratinocytes proliferation related protein K14, which revealed that there was certain similarity in the mRNA expression between keratinocytes and amnion epithelial cells, and indicating that it has the potential to differentiate into keratinocytes. Tissue-engineered skin was successful y constructed by using amniotic mesenchymal stem cells and amnion epithelial cells, hematoxylin-eosin staining section showed that it has certain skin structure, and amnion epithelial cells had a preliminary differentiation. Al these prove that it is feasible to construct human skin tissues with amniotic mesenchymal stem cells and amniotic epithelial cells through the three-dimensional culture.