CAJ | 학술논문

背景:课题组前期实验已证实人羊膜上皮细胞条件培养液可以诱导人脐血间充质干细胞分化为多巴胺能神经元样细胞,在此过程中人羊膜上皮细胞分泌的神经营养因子及其受体可能起到了重要作用.目的:探讨人羊膜上皮细胞分泌的神经营养因子对人脐血间充质干细胞神经分化的作用.方法:将P1代人脐血间充质干细胞按2×10~8 L~(-1)密度接种,分为3组:对照组加入HG-DMEM培养基;诱导组加入人羊膜上皮细胞条件培养液;阻断剂组预先加入阻断剂K252a工作液,36 ℃孵育40 min后更换为羊膜上皮细胞条件培养液.免疫荧光化学检测诱导后人脐血间充质干细胞神经元特异性烯醇化酶及多巴胺转运体的表达,实时定量PCR法检测诱导后人脐血间充质干细胞中神经元特异性烯醇化酶、多巴胺转运体及酪氨酸羟化酶的表达.结果与结论:人羊膜上清中有神经生长因子和脑源性神经营养因子的表达,且P1代人脐血间充质干细胞表达神经营养因子高黏附性受体Trka及Trkb.诱导48 h后与对照组比较,诱导组及阻断剂组神经元特异性烯醇化酶、多巴胺转运体阳性细胞数均明显增加(P < 0.05),且诱导组阳性细胞数最多(P < 0.05).诱导组、阻断剂组神经元特异性烯醇化酶、多巴胺转运体及酪氨酸羟化酶mRNA含量均显著高于对照组(P < 0.01),且诱导组各基因mRNA含量明显高于阻断剂组(P < 0.01).结果证实人羊膜上皮细胞分泌的神经营养因子对人脐血间充质干细胞的神经分化有重要作用,其促神经分化作用是通过酪氨酸激酶受体介导的.
배경:과제조전기실험이증실인양막상피세포조건배양액가이유도인제혈간충질간세포분화위다파알능신경원양세포,재차과정중인양막상피세포분비적신경영양인자급기수체가능기도료중요작용.목적:탐토인양막상피세포분비적신경영양인자대인제혈간충질간세포신경분화적작용.방법:장P1대인제혈간충질간세포안2×10~8 L~(-1)밀도접충,분위3조:대조조가입HG-DMEM배양기;유도조가입인양막상피세포조건배양액;조단제조예선가입조단제K252a공작액,36 ℃부육40 min후경환위양막상피세포조건배양액.면역형광화학검측유도후인제혈간충질간세포신경원특이성희순화매급다파알전운체적표체,실시정량PCR법검측유도후인제혈간충질간세포중신경원특이성희순화매、다파알전운체급락안산간화매적표체.결과여결론:인양막상청중유신경생장인자화뇌원성신경영양인자적표체,차P1대인제혈간충질간세포표체신경영양인자고점부성수체Trka급Trkb.유도48 h후여대조조비교,유도조급조단제조신경원특이성희순화매、다파알전운체양성세포수균명현증가(P < 0.05),차유도조양성세포수최다(P < 0.05).유도조、조단제조신경원특이성희순화매、다파알전운체급락안산간화매mRNA함량균현저고우대조조(P < 0.01),차유도조각기인mRNA함량명현고우조단제조(P < 0.01).결과증실인양막상피세포분비적신경영양인자대인제혈간충질간세포적신경분화유중요작용,기촉신경분화작용시통과락안산격매수체개도적.
BACKGROUND: Group pre-test has confirmed that amnion endothelial cell conditioned medium can induce human umbilical cord blood mesenchymal stem cells into dopaminergic neuron-like cells. In this process, neurotrophic factors and their receptors may play an important role. OBJECTIVE: To study the function of neurotrophic factors secreted by amniotic epithelial cells in the differentiation of human umbilical cord blood mesenchymal stem cells into neurons.METHODS: P1 human umbilical cord blood mesenchymal stem cells at 2×10~8 /L were incubated and assigned to 3 group. Control group was added with HG-DMEM medium. Induction group received human amniotic epithelial cell medium. Blocking agent group underwent blocking agent K252a fluid, and the incubated was conducted at 36 ℃ for 40 minutes, and then amniotic epithelial cell medium was added. Immunofluorescence chemistry was used to determine neuron specific enolase and dopamine transporter expression in human umbilical cord blood mesenchymal stem cells. Real-time quantitative PCR was employed to detect neuron specific enolase, dopamine transporter and tyrosine hydroxylase expression in human umbilical cord blood mesenchymal stem cells. RESULTS AND CONCLUSION: Nerve growth factor and brain-derived neurotrophic factor were observed in human amniotic supernatant. P1 human umbilical cord blood mesenchymal stem cells expressed Trka and Trkb. Forty-eight hours following induction, compared with the control group, positive expression of neuron specific enolase and dopamine transporter was significantly increased in the induction and blocking agent groups (P < 0.05), especially in the induction group (P < 0.05). Neuron specific enolase, dopamine transporter and tyrosine hydroxylase mRNA levels were significantly greater in the induction and blocking agent groups compared with the control group (P < 0.01), and each gene mRNA levels were significantly greater in the induction group than in the blocking agent group (P < 0.01). Results verified that neurotrophic factor in the human amniotic epithelial cells plays important effects on differentiation of human umbilical cord blood mesenchymal stem cells into neurons. The promotion effects are mediated by activating Trk receptor.