基础医学与临床 ›› 2022, Vol. 42 ›› Issue (1): 75-81.

• 研究论文 • 上一篇    下一篇

PF-127-LV-NTFs三维复合支架培养大鼠神经干细胞

梁国新1,梁茵茹2,刘淑贤3,陈康振2,林勇1,秦江霞3,吴洪福2   

  1. 1. 广东医科大学附属第三医院(顺德区龙江医院)
    2. 广东医科大学广东医科大学干细胞与再生组织工程重点实验室
    3. 广东医科大学附属广州花都医院(广州市花都区妇幼保健院)
  • 收稿日期:2021-09-27 修回日期:2021-11-19 出版日期:2022-01-05 发布日期:2022-01-05
  • 通讯作者: 吴洪福 E-mail:hongfuw@126.com
  • 基金资助:
    长链非编码RNA Vof-16调控脊髓内源性神经干细胞参与脊髓损伤修复的机制研究;长链非编码RNA Vof-16在脊髓损伤中的作用和分子机制研究;长链非编码RNA Vof-16调控脊髓损伤神经修复的作用和机制研究; hucMSC-PRP-水凝胶组织工程成骨支架构建及其修复关节软骨损伤的研究;LncRNA Vof-16干预脊髓损伤后神经再生的作用机制研究

PF-127-LV-NTFs three-dimensional composite scaffold in culturing rat neural stem cells

  • Received:2021-09-27 Revised:2021-11-19 Online:2022-01-05 Published:2022-01-05
  • Contact: Hong-Fu WU E-mail:hongfuw@126.com

摘要: 目的 观察Pluronic F-127(PF-127)水凝胶负载编码慢病毒LINGO-1 shRNA(LV)及神经生长因子混合物(NF, NFcocktail)三维复合支架对大鼠神经干细胞(NSCs)增殖分化的影响。方法 构建LV后以不同感染复数(MOI)感染NSCs,用RT-qPCR及Western blot检测NSCs中LINGO-1的表达;按照一定比例将脑源性神经营养因子(BDNF)、神经营养因子-3(NT-3)、血小板衍生生长因子(PDGF)、胰岛素样生长因子-1(IGF-1)、表皮生长因子(EGF)、碱性成纤维细胞生长因子(bFGF)、神经胶质细胞系来源的神经营养因子(GDNF)配制成NFcocktail,PF-127水凝胶负载LV和NFcocktail并分组培养NSCs;用CCK-8法检测NSCs的细胞活力,LIVE/DEAD染色检测NSCs的存活率,免疫荧光染色检测NSCs增殖和分化情况。结果 LV感染NSCs的最适MOI=5;PF-127对NSCs的细胞活力没有影响;PF-127+LV+NF组NSCs存活、增殖及神经元分化比例较高(P<0.01)。结论 Pluronic F-127水凝胶/LINGO-1 shRNA/神经生长因子混合物(PF-127-LV-NFcocktail)三维复合支架有利于大鼠NSCs的增殖和神经元的定向分化。

关键词: NSCs, LINGO-1 shRNA, PF-127水凝胶, 神经元分化

Abstract: Objective To investigate the effect of LINGO-1 shRNA (LV) and neurotrophic factors cocktail (NF,NFcocktail) encapsulated by Pluronic F-127(PF-127)hydrogel three-dimensional composite scaffolds on proliferation and differentiation of rat neural stem cells. Methods The LV was constructed and infected with NSCs at different MOI. RT-qPCR and Western blot were used to detect the expression of LINGO-1 in NSCs. BDNF NT-3 PDGF IGF-1 EGF bFGF and GDNF groups were divided into NFcocktail. The LV and NFcocktail were mixed with PF-127 and divided into different groups to culture NSCs. CCK-8 was used to detect the of cell viability of NSCs, the survival rate of NSCs was detected by LIVE/DEAD staining, and immunofluorescence staining was used to detect the proliferation and differentiation of NSCs. Results the optimal MOI for LV was 5. CCK-8 assay results showed that there was no significant difference in the survival rate of NSCs. The LIVE/DEAD and Nestin immune-fluorescence staining results showed that compared with control, the survival rate of NSCs and the proportion of neuron differentiation positive were the highest in PF-127+LV+NF group(P<0.01). Conclusions PF-127-LV-NFcocktail three-dimensional composite scaffold is beneficial to the proliferation and the directional differentiation of NSCs in rat.

Key words: neural stem cells, LINGO-1 shRNAl, PF-127 hydrogel, neuronal differentiation

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