CAJ | 학술논문

目的观察猪脂肪来源干细胞的分离、体外培养方法及基本生物学特性,探讨其多向诱导分化潜能.方法取猪颈部皮下脂肪组织,酶消化法获得脂肪干细胞,细胞免疫荧光及流式细胞技术检测细胞表面抗原,诱导其向脂肪细胞、软骨细胞和成骨细胞分化,油红染色、逆转录-聚合酶链反应(RT-PCR)和茜素红染色检测细胞诱导分化.结果成功培养出脂肪干细胞,流式细胞术检测CD44、CD105的阳性率分别为90.2%和99.2%,CD34、CD45的阳性率分别为0.3%和1.2%.成脂肪诱导3周后经油红O染色显示细胞内有脂滴形成,诱导成功率为93%,RT-PCR检测显示细胞能表达过氧化物酶体增殖物激活受体(PPAR-γ);成软骨诱导4周后,RT-PCR检测结果显示细胞能表达软骨聚集蛋白聚糖(aggrecan)和Ⅱ型胶原;成骨诱导3周后,茜素红染色发现细胞出现钙结节,诱导成功率为94%.结论猪脂肪干细胞在体外具有向成脂肪、成软骨和成骨细胞分化的潜能,有望成为组织工程较理想的种子细胞之一.
목적 관찰저지방래원간세포적분리、체외배양방법 급기본생물학특성,탐토기다향유도분화잠능.방법 취저경부피하지방조직,매소화법획득지방간세포,세포면역형광급류식세포기술검측세포표면항원,유도기향지방세포、연골세포화성골세포분화,유홍염색、역전록-취합매련반응(RT-PCR)화천소홍염색검측세포유도분화.결과 성공배양출지방간세포,류식세포술검측CD44、CD105적양성솔분별위90.2%화99.2%,CD34、CD45적양성솔분별위0.3%화1.2%.성지방유도3주후경유홍O염색현시세포내유지적형성,유도성공솔위93%,RT-PCR검측현시세포능표체과양화물매체증식물격활수체(PPAR-γ);성연골유도4주후,RT-PCR검측결과 현시세포능표체연골취집단백취당(aggrecan)화Ⅱ형효원;성골유도3주후,천소홍염색발현세포출현개결절,유도성공솔위94%.결론 저지방간세포재체외구유향성지방、성연골화성골세포분화적잠능,유망성위조직공정교이상적충자세포지일.
Objective To investigate the isolation, culture, biological characteristics and multi-differentiation of porcine adipose-derived stem cells (ADSCs) in vitro. Methods ADSCs were obtained from back subcutaneous adipose tissue of porcine by collagenase digestion and cultured in vitro. Flow cytometric analysis and cyto-immumofluorescence staining technique were carried out to detect the immunophenotypes of ADSCs. Reverse transcription-polymerase chain reaction (RT-PCR) analysis and specific staining were used to evaluate the multipotential differentiation phenotype of ADSCs. Results The stem cells isolated from subcutaneous adipose tissue were positive for CD44 (90.2%) and CD105 (99.2%), lacking in CD34 (0.3%) and CD45 (1.2%). After culture in mesenchymal stem cell adipogenic, chondrogenic and osteogenic differentiation basal media respectively for 3, 4 and 3 weeks, the cells were positive for oil red O staining (93%) and alizarin bordeaux staining (94%), also the expression of peroxisome proliferator activated receptor (PPAR)-γ, collagen and aggrecan was detected in the differentiated cells. Conclusion The porcine ADSCs can differentiate towards adipocytes, chondrocytes and osteoblasts when culture in certain medium, and ADSCs can likely served as one of the most optimal autogenous cell sources for tissue engineering.