皮肤源性iPS的制备及其定向神经分化机制的研究

发布时间：2018-05-23 15:18

  本文选题：皮肤干细胞 + Ngn2　； 参考：《南京医科大学》2011年硕士论文

【摘要】：研究目的 探讨皮肤源性诱导式多潜能干细胞(induced pluripotent stem iPS)的制备及体外定向神经分化的机制。 研究方法 1.体外培养大鼠皮肤来源前体细胞(skin-derived precursors SKPs)并纯化、鉴定。 2.构建包装含Ngn2基因质粒的慢病毒载体并用其转染第3代SKPs。 3.SKPs分三组:A组为慢病毒载体介导Neurogenin2(Ngn2)基因转染的SKps,B组为空载体慢病毒转染的SKps,C组为未经处理的的SKPs。 4.诱导7天后,免疫荧光检测各组细胞神经分化效率的差异。 5.Western Blot检测各组细胞神经分化过程中Notch信号通路相关蛋白hes1和Dll1表达水平的差异。 研究结果 1.皮肤源性iPS可持续稳定传代并表达绿色荧光。 2.诱导培养基诱导7天后,免疫荧光结果显示A组神经元标志蛋白阳性细胞率均明显高于B组和C组,结果有均显著统计学差异(P0.05)。 3.诱导7天后,A组细胞Dll1蛋白的表达水平明显高于B组和C组,而Hes1蛋白的表达水平明显显低于B组和C组,结果均有显著统计学差异(P0.01)。 研究结论 1.慢病毒介导Ngn2基因转染SKPs,通过细胞程序重排技术,可制备出皮肤源性iPS。 2.皮肤源性iPS向神经细胞定向分化的效率明显高于SKPs。 3.皮肤源性iPS高神经分化的机制可能通过上调Dll1蛋白水平,下调hes1蛋白水平,抑制了Notch信号通路,从而促进神经分化。
[Abstract]:Research purpose To investigate the preparation of pluripotent stem iPS) induced by skin induced multipotential stem cells and the mechanism of directional neural differentiation in vitro. Research method 1. Rat skin derived progenitor cells (skin-derived precursors SKPs) were cultured in vitro and purified and identified. 2. The lentivirus vector containing Ngn2 gene plasmid was constructed and transfected into SKPs3. 3.SKPs was divided into three groups: group A: lentivirus vector mediated neurogenin2ngn2) gene transfection. Group B was transfected with empty vector lentivirus. Group C was untreated SKPs. 4. After 7 days of induction, the difference of neural differentiation efficiency was detected by immunofluorescence. 5.Western Blot was used to detect the expression of Notch signaling pathway related protein hes1 and Dll1 in the process of neural differentiation. Research results 1. Skin derived iPS can be continuously and stably passaged and expressed green fluorescence. 2. The results of immunofluorescence showed that the positive rate of neuronal marker protein in group A was significantly higher than that in group B and C, and there was significant difference between group A and group C (P 0.05). 3. After 7 days of induction, the expression of Dll1 protein in group A was significantly higher than that in group B and group C, while the expression level of Hes1 protein in group A was significantly lower than that in group B and group C (P 0.01). Research conclusion 1. Ngn2 gene was transfected into SKPsby lentivirus, and skin-derived iPSs could be prepared by cell program rearrangement. 2. The differentiation efficiency of skin derived iPS into neural cells was significantly higher than that of SKPs. 3. The mechanism of high neural differentiation in dermatogenic iPS may promote neural differentiation by upregulating the level of Dll1 protein and down-regulating the level of hes1 protein, thus inhibiting the Notch signaling pathway.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

【参考文献】

相关期刊论文 前2条

1 李立新,吴幼章,胡卫星,顾培元,傅震,徐启武;甲基强的松龙和神经干细胞移植联合治疗大鼠脊髓损伤[J];中华神经外科疾病研究杂志;2002年03期

2 李立新,徐启武,车晓明;神经干细胞移植促进鼠脊髓损伤后髓鞘结构的修复[J];中国神经精神疾病杂志;2002年06期

，

本文编号：1925300