中国组织工程研究
中國組織工程研究
중국조직공정연구
Journal of Clinical Rehabilitative Tissue Engineering Research
2014年
23期
3682-3690
,共9页
周文然%李新%王文波%谢燕霞%唐娜%阎影
週文然%李新%王文波%謝燕霞%唐娜%閻影
주문연%리신%왕문파%사연하%당나%염영
干细胞%培养%分化%羊膜%间充质干细胞%羊膜上皮细胞%诱导分化%多巴胺能神经元
榦細胞%培養%分化%羊膜%間充質榦細胞%羊膜上皮細胞%誘導分化%多巴胺能神經元
간세포%배양%분화%양막%간충질간세포%양막상피세포%유도분화%다파알능신경원
stem cells%amnion%mesenchymal stem cells%epithelial cells%celldifferentiation%dopamine%neurons
背景：羊膜间充质干细胞具有类似胚胎干细胞多潜能的特点，在再生医学等多种领域的临床应用具有广泛的实用性和明确的良好前景。然而，目前对于羊膜间充质干细胞的生物学特性和分化潜能的认识仍然了解甚少。目的：建立体外分离和纯化人羊膜间充质干细胞的方法，检测羊膜间充质干细胞的体外分化特点，确定羊膜间充质干细胞在体外诱导条件下向多巴胺能神经元样细胞分化的潜能。方法：采用胰蛋白酶和胶原酶Ⅱ分步消化法从羊膜中分离羊膜间充质干细胞和羊膜上皮细胞；采用percol 梯度离心方法对羊膜间充质干细胞和羊膜上皮细胞进行纯化；流式细胞术检测羊膜间充质干细胞的表面标志，确定羊膜间充质干细胞细胞表面抗原的表达特征；对体外培养的羊膜间充质干细胞进行成脂肪和成骨诱导，确定其多向分化的潜能；采用神经细胞条件培养体系诱导羊膜间充质干细胞向多巴胺神经细胞分化，通过免疫荧光染色和激光共聚焦荧光显微镜观察和鉴定诱导后多巴胺神经元样细胞的生成。结果与结论：从羊膜组织成功分离、纯化和培养出羊膜间充质干细胞和羊膜上皮细胞。羊膜来源的间充质干细胞不仅具有典型的间充质干细胞标志，而且保留了一些胚胎干细胞的OCT-4，SOX-2和KLF4等特有干细胞标志，可以诱导分化成为脂肪细胞和骨细胞，显示羊膜间充质干细胞保持了较为原始的胚胎干细胞的特点，具有多向分化的潜能。诱导分化之前的原代羊膜间充质干细胞表达固有的多种神经细胞标记，体外诱导后羊膜间充质干细胞可分化成为β-微管蛋白Ⅲ、神经元特异性核蛋白、酪氨酸羟化酶、胶质纤维酸性蛋白、髓鞘碱性蛋白和巢蛋白等阳性表达的多巴胺能神经元样细胞。结果表明人羊膜来源的间充质干细胞所保留的多向分化潜能和有效分化成为多巴胺能神经元样细胞的特性。
揹景：羊膜間充質榦細胞具有類似胚胎榦細胞多潛能的特點，在再生醫學等多種領域的臨床應用具有廣汎的實用性和明確的良好前景。然而，目前對于羊膜間充質榦細胞的生物學特性和分化潛能的認識仍然瞭解甚少。目的：建立體外分離和純化人羊膜間充質榦細胞的方法，檢測羊膜間充質榦細胞的體外分化特點，確定羊膜間充質榦細胞在體外誘導條件下嚮多巴胺能神經元樣細胞分化的潛能。方法：採用胰蛋白酶和膠原酶Ⅱ分步消化法從羊膜中分離羊膜間充質榦細胞和羊膜上皮細胞；採用percol 梯度離心方法對羊膜間充質榦細胞和羊膜上皮細胞進行純化；流式細胞術檢測羊膜間充質榦細胞的錶麵標誌，確定羊膜間充質榦細胞細胞錶麵抗原的錶達特徵；對體外培養的羊膜間充質榦細胞進行成脂肪和成骨誘導，確定其多嚮分化的潛能；採用神經細胞條件培養體繫誘導羊膜間充質榦細胞嚮多巴胺神經細胞分化，通過免疫熒光染色和激光共聚焦熒光顯微鏡觀察和鑒定誘導後多巴胺神經元樣細胞的生成。結果與結論：從羊膜組織成功分離、純化和培養齣羊膜間充質榦細胞和羊膜上皮細胞。羊膜來源的間充質榦細胞不僅具有典型的間充質榦細胞標誌，而且保留瞭一些胚胎榦細胞的OCT-4，SOX-2和KLF4等特有榦細胞標誌，可以誘導分化成為脂肪細胞和骨細胞，顯示羊膜間充質榦細胞保持瞭較為原始的胚胎榦細胞的特點，具有多嚮分化的潛能。誘導分化之前的原代羊膜間充質榦細胞錶達固有的多種神經細胞標記，體外誘導後羊膜間充質榦細胞可分化成為β-微管蛋白Ⅲ、神經元特異性覈蛋白、酪氨痠羥化酶、膠質纖維痠性蛋白、髓鞘堿性蛋白和巢蛋白等暘性錶達的多巴胺能神經元樣細胞。結果錶明人羊膜來源的間充質榦細胞所保留的多嚮分化潛能和有效分化成為多巴胺能神經元樣細胞的特性。
배경：양막간충질간세포구유유사배태간세포다잠능적특점，재재생의학등다충영역적림상응용구유엄범적실용성화명학적량호전경。연이，목전대우양막간충질간세포적생물학특성화분화잠능적인식잉연료해심소。목적：건입체외분리화순화인양막간충질간세포적방법，검측양막간충질간세포적체외분화특점，학정양막간충질간세포재체외유도조건하향다파알능신경원양세포분화적잠능。방법：채용이단백매화효원매Ⅱ분보소화법종양막중분리양막간충질간세포화양막상피세포；채용percol 제도리심방법대양막간충질간세포화양막상피세포진행순화；류식세포술검측양막간충질간세포적표면표지，학정양막간충질간세포세포표면항원적표체특정；대체외배양적양막간충질간세포진행성지방화성골유도，학정기다향분화적잠능；채용신경세포조건배양체계유도양막간충질간세포향다파알신경세포분화，통과면역형광염색화격광공취초형광현미경관찰화감정유도후다파알신경원양세포적생성。결과여결론：종양막조직성공분리、순화화배양출양막간충질간세포화양막상피세포。양막래원적간충질간세포불부구유전형적간충질간세포표지，이차보류료일사배태간세포적OCT-4，SOX-2화KLF4등특유간세포표지，가이유도분화성위지방세포화골세포，현시양막간충질간세포보지료교위원시적배태간세포적특점，구유다향분화적잠능。유도분화지전적원대양막간충질간세포표체고유적다충신경세포표기，체외유도후양막간충질간세포가분화성위β-미관단백Ⅲ、신경원특이성핵단백、락안산간화매、효질섬유산성단백、수초감성단백화소단백등양성표체적다파알능신경원양세포。결과표명인양막래원적간충질간세포소보류적다향분화잠능화유효분화성위다파알능신경원양세포적특성。
BACKGROUND:Human amniotic membrane-derived mesenchymal stem cells (AMSCs) are considered to be one kind of adult stem cells that can be easily obtained in large quantities without using an invasive method. Because of their low immunogenicity, anti-inflammatory properties, multipotency of differentiation and without ethical issue, human amniotic membrane-derived mesenchymal stem cells have been proposed as a good candidate to be used in celltherapy and regenerative medicine. However, the biological properties and the differentiation capacity of human amniotic membrane-derived mesenchymal stem cells are stil poorly characterized. OBJECTIVE:To establish a practical method for isolation and purification of human amniotic membrane-derived mesenchymal stem cells, and to study the biological characteristics and dopaminergic neural-like celldifferentiation potential of the human amniotic membrane-derived mesenchymal stem cells. METHODS:Human amniotic membrane-derived mesenchymal stem cells were disassociated and isolated from the amniotic membrane by trypsin and col agenase based enzymic digestion, and purified by percol mediated density gradient centrifugation. Expressions of surface antigens and transcription factors of the human amniotic membrane-derived mesenchymal stem cells were determined by flow cytometry and western blot assays. Based on the osteogenic and adipogenic induction, the multipotent differentiation capability of human amniotic membrane-derived mesenchymal stem cells was determined. Induction of neural celldifferentiation of human amniotic membrane-derived mesenchymal stem cells was conducted in Neurabasal conditioning medium with ATRA supplement. Neural cellassociated bio-markers were determined by immunofluoresence staining and confocal microscope. RESULTS AND CONCLUSION:In this study, we performed a practical method to isolate and purify human amniotic membrane-derived mesenchymal stem cells and amniotic epithelial cells simultaneously, with high cells yield. We demonstrated a group of constitutive expressions of neural antigens and embryonic associated transcription factor proteins (OCT-4, SOX-2 and KLF4) in fresh isolated human amniotic membrane-derived mesenchymal stem cells as wel as in human amniotic membrane-derived mesenchymal stem cells after in vitro passage, which suggested that the human amniotic membrane-derived mesenchymal stem cells not only possessed intrinsic tendency to neural celldifferentiation, but also maintained their stem cellcharacteristics after in vitro passage. We stimulated the human amniotic membrane-derived mesenchymal stem cells in the neurobasal-A and B27 based conditioning medium to induce neural celldifferentiation. The induced human amniotic membrane-derived mesenchymal stem cells displayed an up-regulation of expression in panel of neural and dopaminergic associate molecules (β-tubulin III, neuron-specific nuclear protein, tyrosine hydroxylase, glial fibril ary acidic protein, myelin basic protein and nestin) by flow cytometry and immunofluorescence staining, which demonstrated the multipotent differentiation capability and dopaminergic neuron-like differentiation potential of the human amniotic membrane-derived mesenchymal stem cells.
