碱性成纤维细胞生长因子和Noggin诱导人羊水来源干细胞向神经细胞分化的差异比较

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.

Differences between Noggin and basic fibroblast growth factor in differentiation of amniotic fluid derived stem cells into nerve cells

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.