CAJ | 학술논문

目的观察体外分离、培养的兔羊膜成体干细胞(ADSC)定向诱导神经细胞的分化结果.方法新西兰雌兔羊膜分离、培养ADSC.实时定量聚合酶链反应(PCR)检测羊膜ADSC的纤连蛋白(Fibronectin)、巢蛋白(Nestin)、波形蛋白(Vimentin) mRNA的表达水平.单克隆形成实验证实羊膜ADSC是否具有自我复制能力.培养成功的ADSC中加入神经分化诱导培养基诱导神经细胞分化.诱导分化5d后,免疫荧光染色检测β-微管蛋白(tubulin)和胶质纤维酸性蛋白(GFAP)的表达.结果培养的ADSC在24 h后逐渐从羊膜组织中移行出；72 h可见明显的多角形细胞富集在羊膜组织周边；120 h后多角形细胞致密地分布于羊膜四周.分裂后的子代细胞形态同父代细胞.单克隆形成实验结果证实羊膜ADSC具有自我复制能力.实时定量PCR检测结果显示,羊膜ADSC的Nestin、Vimentin、Fibronectin mRNA分别是脾脏细胞同种分子表达量的15.79、1.91、7.65倍,两者差异有统计学意义(Z=-5.243、-3.972、-2.524,P＜0.05).免疫荧光染色结果显示,β-tubulin在大部分细胞的胞质中表达；GFAP在部分细胞的细胞质中表达.结论羊膜ADSC具有自我复制能力,在合适条件下能够分化成神经元和神经胶质细胞.
목적 관찰체외분리、배양적토양막성체간세포(ADSC)정향유도신경세포적분화결과.방법 신서란자토양막분리、배양ADSC.실시정량취합매련반응(PCR)검측양막ADSC적섬련단백(Fibronectin)、소단백(Nestin)、파형단백(Vimentin) mRNA적표체수평.단극륭형성실험증실양막ADSC시부구유자아복제능력.배양성공적ADSC중가입신경분화유도배양기유도신경세포분화.유도분화5d후,면역형광염색검측β-미관단백(tubulin)화효질섬유산성단백(GFAP)적표체.결과 배양적ADSC재24 h후축점종양막조직중이행출；72 h가견명현적다각형세포부집재양막조직주변；120 h후다각형세포치밀지분포우양막사주.분렬후적자대세포형태동부대세포.단극륭형성실험결과증실양막ADSC구유자아복제능력.실시정량PCR검측결과현시,양막ADSC적Nestin、Vimentin、Fibronectin mRNA분별시비장세포동충분자표체량적15.79、1.91、7.65배,량자차이유통계학의의(Z=-5.243、-3.972、-2.524,P＜0.05).면역형광염색결과현시,β-tubulin재대부분세포적포질중표체；GFAP재부분세포적세포질중표체.결론 양막ADSC구유자아복제능력,재합괄조건하능구분화성신경원화신경효질세포.
Objective To observe the differentiation effect of rabbit amnion-derived stem cells (ADSC) induced into neural cells.Methods ADSC of New Zealand female rabbits were isolated and cultured.Its mRNA level of Fibronectin,Nestin and Vimentin were detected by real-time quantitative polymerase chain reaction.The self-replication ability of ADSC was confirmed by monoclonal formation experiments.These ADSC were further induced into neural cells in vitro.Five days after induced differentiation,the expression of β-tubulin and glial fibrillary acidic protein (GFAP) were detected by immunofluorescent staining.Results ADSC were separated from amnion tissue gradually after 24 hours.There were polygonal cells gathered around the amnion tissue at 72 hours,and were distributed compactly around the amnion at 120 hours.The morphology of cleavage daughter cells was basically the same as parent cells.ADSC has the ability of self-replication.The Nestin,Vimentin,Fibronectin mRNA expressions in ADSC were 15.79,1.91,7.65 times those in spleen cells.The differences were statistically significant (Z=-5.243,-3.972,-2.524; P＜0.05).The β-tubulin expression was found in cytoplasm of most cells.The GFAP expression was found in cytoplasm in some cells.Conclusions ADSC has self-replication ability.It can be induced into neurons and neuroglial cells under the right conditions.