摘要
背景:近年来,在啮齿类动物中发现,造血干细胞具有向神经分化的塑型性,而在人类,这种造血的塑型性仍然具有争议。目的:检测人脐血来源的AC133+造血干细胞是否具有向神经分化的潜能。设计、时间及地点:本实验为成组对照设计实验,于2005-08/2007-12在天津血液学研究所和清华大学玉泉医院神经中心实验室完成。材料:人脐带取自健康足月新生儿。胎脑滋养层细胞来源于22周流产胎儿脑组织。方法:应用免疫磁珠分选人脐血AC133+细胞,流式细胞仪检测其纯度为99%以上。同时以机械分离及酶消化法制备胎脑滋养层细胞。选用4种培养条件对脐血来源AC133+造血干细胞进行诱导分化:①生长培养液DMEM/F12加上皮生长因子和碱性成纤维生长因子。②DMEM/F12加上皮生长因子和碱性成纤维生长因子并联合使用脑源性生长因子,其间用全反式维甲酸处理3d。③非细胞接触的共培养体系,先将制备的胎脑滋养层细胞生长在培养板内插件的半透膜上,与培养板内的脐血来源的AC133+细胞进行共培养。④细胞直接接触共培养,将预先用BrdU标记的脐血来源的AC133+细胞直接种在胎脑滋养层细胞上进行共培养。主要观察指标:1,2,4周收集诱导分化的脐血细胞,以RT-PCT检测是否有巢蛋白、骨形态生成蛋白2及神经细胞黏附分子的表达,免疫细胞化学方法检测细胞分型特异性抗原。结果:部分脐血来源的AC133+细胞,在体外存在生长必需的因子上皮生长因子和碱性成纤维生长因子时,能表达一些与神经细胞分化有关的基因,如巢蛋白、骨形态生成蛋白,条件不同时,这些基因出现下调和关闭。在DMEM/F12加上皮生长因子和碱性成纤维生长因子联合使用脑源性生长因子诱导培养液中,上述基因表达在2周时可检测到,同时可检测到神经发育过程中出现的另一分子神经细胞黏附分子,这一分子在造血过程中并不出现,在使用内插件的胎脑滋养层细胞共培养的脐血细胞中也检测到相同结果。在优化的非细胞接触条件中主要表达胶质酸性蛋白,在细胞直接接触的共培养体系中出现神经元分化的标志物Ⅲβ-tubulin蛋白的表达。这种基因表达变化提示,在特定的培养环境下,脐血来源的AC133+细胞内可能出现了基因重排,使造血潜能的干细胞表达神经细胞发育分子。结论:脐血来源的AC133+细胞包含部分具有向神经分化潜能的多能干细胞,在适合条件下,表达神经分化相关抗原。
BACKGROUND： At present, hemopoietic stem cells have been proved to differentiate into nerves in rodents animals. As for the human, this topic is in debate. OBJECTIVE： To investigate the neural differentiation potential of human umbilical cord blood-derived AC 133^＋ cells. DESIGN, TIME AND SETTING： Control experiments by grouping were performed in the Hematology Institute of Tianjin Hematology Hospital and Central Laboratory of Neurosurgery in Yuquan Hospital of Tsinghua University from August 2005 to December 2007. MATERIALS： Human umbilical cord blood was sampled from full-term newborn infant. Fetal brain-derived trophic support cells were harvested from aborted fetus of 22 weeks old. METHODS： Human umbilical cord blood-derived AC 133^＋ cells were screened out by using immunomagnetic beads, and flow cytometry indicated the purity of cells was over 99%. Meanwhile, fetal brain-derived trophic support cells were prepared with mechanical separation and enzyme digestion methods. Four different culture conditions were established to detect the differentiation of AC133^＋ cells. （1）DMEM/F12 medium supplemented with epidermal growth factor and basic fibroblast growth factor. （2） DMEM/F 12 medium supplemented with epidermal growth factor, basic fibroblast growth factor, brain-derived neurotrophic factor and retinoic acid for 3 days. （3）Non cell-cell contact co-culture system： Human cord blood isolated AC133^＋ cells were co-cultured with fetal brain-derived trophic support cells, which were previously inoculated on the semipermeable membrane. （4）Co-culture system with direct cell-cell contact： BrdU-laheled AC 133^＋ cells were directly co-cultured with fetal brain-derived trophic support cells. MAIN OUTCOME MEASURES： After the induction, human cord blood cells were collected at weeks 1, 2 and 4. RT-PCR was used to detect the expression of nestin, bone morphogenetic protein-2 and neural cell adhesion molecule. Immunocytochemistry method was applied to detect the cytotype-specific antigen. RESULTS： In the culture medium containing epidermal growth factor and basic fibroblast growth factor, human cord blood AC 133^＋ cells could express nestin and bone morpbogenetic protein-2, which were down-regulated even closed up in suboptimal condition. In the DMEM/F12 medium supplemented with epidermal growth factor, basic fibroblast growth factor and brain-derived neurotrophic factor, the gene expression of bone morphogenetic protein-2 and nestin continued in optimal condition at 2 weeks. Moreover neural cell adhesion molecule, another gene of neural cells, also expressed in this condition. AC133^＋ cells co-cultured with fetal brain-derived trophic support cells exhibited similar expressions. In the optimal non-cell-cell contact co-culture system, glial fibrillary acidic protein-positive cells were found by immuocytochemistry, while neuronal marker β -tubulin Ⅲ was expressed in the cell-cell direct contact system. These outcomes indicated that human cord blood isolated AC133^＋ cells may have an effect through gene rearrangement on inducing stem cells to express nerve cell development factors. CONCLUSION： The human umbilical cord blood-derived AC 133^＋ cells contain some multipotential stern cells with differentiation potential, neural differentiation-related antigen when exposed to a suitable microenvironment.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2008年第38期7566-7572,共7页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
Yue Yuen Educational Foundation of Tsinghua University, No. 20240000540~~
