CAJ | 학술논문

背景:相对于成体干细胞和胚胎干细胞自身存在的问题,羊水来源的干细胞系的建立,能为每一个个体建立一份自身的、具有高度增殖分化能力的干细胞储备,有望成为神经性退行性疾病细胞治疗的理想细胞来源.目的:观察Noggin和碱性成纤维细胞生长因子对人羊水来源干细胞向神经细胞分化的影响.方法:羊水标本来自怀孕16-22周行产前诊断的孕妇,在超声引导下行羊膜腔穿刺取得.利用CD117抗体,选用免疫磁珠法从人孕中期羊水标本中分离获得羊水干细胞,培养扩增后,通过流式细胞仪检测表面抗原表达进行鉴定.选取生长状态良好的第3代羊水干细胞,利用无血清的神经诱导培养液诱导其向神经细胞分化,分为空白对照组、基础诱导液组、Noggin诱导组和碱性成纤维细胞生长因子诱导组.倒置相差显微镜观察诱导后细胞形态的变化,细胞免疫荧光检测巢蛋白、β-Ⅲtubulin和神经丝蛋白在诱导后细胞中的表达.结果与结论:利用免疫磁珠方法分离出的羊水干细胞呈CD44和HLA-ABC表达阳性,CD45和HLA-DR表达阴性.诱导2周后,碱性成纤维细胞生长因子诱导组光镜下细胞形态发生显著变化,免疫荧光染色巢蛋白、β-Ⅲ tubulin和神经丝蛋白表达阳性率较高.Noggin诱导组细胞形态和染色结果与基础诱导液组无显著性差异.提示利用羊水来源的干细胞诱导分化为神经细胞的过程中碱性成纤维细胞生长因子的作用远优于Noggin.
배경:상대우성체간세포화배태간세포자신존재적문제,양수래원적간세포계적건립,능위매일개개체건립일빈자신적、구유고도증식분화능력적간세포저비,유망성위신경성퇴행성질병세포치료적이상세포래원.목적:관찰Noggin화감성성섬유세포생장인자대인양수래원간세포향신경세포분화적영향.방법:양수표본래자부잉16-22주행산전진단적잉부,재초성인도하행양막강천자취득.이용CD117항체,선용면역자주법종인잉중기양수표본중분리획득양수간세포,배양확증후,통과류식세포의검측표면항원표체진행감정.선취생장상태량호적제3대양수간세포,이용무혈청적신경유도배양액유도기향신경세포분화,분위공백대조조、기출유도액조、Noggin유도조화감성성섬유세포생장인자유도조.도치상차현미경관찰유도후세포형태적변화,세포면역형광검측소단백、β-Ⅲtubulin화신경사단백재유도후세포중적표체.결과여결론:이용면역자주방법분리출적양수간세포정CD44화HLA-ABC표체양성,CD45화HLA-DR표체음성.유도2주후,감성성섬유세포생장인자유도조광경하세포형태발생현저변화,면역형광염색소단백、β-Ⅲ tubulin화신경사단백표체양성솔교고.Noggin유도조세포형태화염색결과여기출유도액조무현저성차이.제시이용양수래원적간세포유도분화위신경세포적과정중감성성섬유세포생장인자적작용원우우Noggin.
BACKGROUND: The establishment of amniotic fluid derived stem cells (AFS) can provide an individual reserve for cell therapy in nerve degenerative diseases.OBJECTIVE: To observe the effects of Noggin and basic fibroblast growth factor (bFGF) on AFS differentiation into neural cells.METHODS: Samples of amniotic fluid were obtained through amniocentesis by ultrasound from gestational age of 16-22 weeks for routine prenatal diagnosis. AFS were obtained from the 2~(nd) trimester amniotic fluid samples by immunomagnetic beads selection using CD117 antibody, and identified the surface antigen expression by flow cytometry after amplification. The 3~(rd) generation of AFS with good growth state were induced to differentiate into nerve cells, which were divided into the blank control,based-induced, Noggin-induced and bFGF-induced groups. The induced cell morphology was observed under inverted phase contrast microscopy, and the expression of nestin, β-Ⅲ tubulin and neurofilament in the induced cells was measured by using cell immunofluorescence detection.RESULTS AND CONCLUSION: Flow cytometry analysis indicated that most of AFS cells expressed CD44 and HLA-ABC, but negative for CD45 and HLA-DR. At 2 weeks after induction, the cell morphology exhibited significant changes with increased Nestin,β-Ⅲ tubulin and NF-positive rates in the bFGF-induced group. However, it had no significant difference in the Noggin-induced group and the based-induced group. It revealed that bFGF plays a vital role in the AFS differentiated into nerve cells.