PF-127-LV-NTFs三维复合支架培养大鼠神经干细胞

doi:10.16352/j.issn.1001-6325.2022.01.008

基础医学与临床 ›› 2022, Vol. 42 ›› Issue (1): 75-81.doi: 10.16352/j.issn.1001-6325.2022.01.008

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^2,3#, 刘淑贤², 陈康振³, 林勇¹, 秦江霞^2*, 吴洪福^3*

1.广东医科大学附属第三医院(顺德龙江医院), 广东佛山 528318;
2.广东医科大学附属广州花都医院,广东广州 510800;
3.广东医科大学干细胞与再生组织工程重点实验室, 广东东莞 523808

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: ^* hongfuw@126.com; qinjx03@163.com
作者简介:^#对本文有相同贡献
基金资助:
国家自然科学基金(82071374);广东省普通高校特色创新项目(2018KTSCX075);东莞市社会发展重点项目(20185071521640);广东医科大学学科建设类二级项目(1.13);广东省医学科学技术研究基金(A2020589)

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

LIANG Guo-xin^1#, LIANG Yin-ru^2,3#, LIU Shu-xian², CHEN Kang-zhen³, LIN Yong¹, QIN Jiang-xia^2*, WU Hong-fu^3*

1. The Third Affiliated Hospital, Guangdong Medical University (Shunde Longjiang Hospital),Foshan 528318;
2. Guangzhou Huadu Hospital, Guangdong Medical University,Guangzhou 510800;
3. Key Laboratory of Stem Cell and Regenerative Tissue Engineering,Guangdong Medical University, Dongguan 523808, China

Received:2021-09-27 Revised:2021-11-19 Online:2022-01-05 Published:2022-01-05
Contact: ^* hongfuw@126.com; qinjx03@163.com

摘要/Abstract

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)及神经营养因子混合物(NTFs)(PF-127-LV-NTFs)三维复合支架对大鼠神经干细胞(NSCs)增殖分化的影响。方法构建慢病毒(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)、神经营养素-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)、神经胶质细胞来源的神经营养因子(GDNF)配制成NTFcocktail(NTFs),PF-127水凝胶负载LV和NTFcocktail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+NTFs组NSCs存活、增殖及神经元分化比例较高(P＜0.01)。结论 PF-127-LV-NTFs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: 神经干细胞, 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 (NTFs,NTFcocktail) encapsulated by Pluronic F-127(PF-127)hydrogel (PF-127-LV-NTFs)three-dimensional(3D) composite scaffolds on the 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. Brain derived neurotropic factor(BDNF), neurotrophin-3(NT-3), platelet-derived growth factor(PDGF),insulin-like growth factor 1(IGF-1), epidermat growth factor(EGF), basic fibroblast growth factor(bFGF) and glial cell derived neurotrophic factor(GDNF) were prepared as NTFs. The LV and NTFs were mixed with PF-127 and divided into different groups to culture NSCs. CCK-8 was used to detect the 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+NTF group(P＜0.01). Conclusions PF-127-LV-NTFs 3D composite scaffold is favouring to support the proliferation and the directional differentiation of NSCs of rat.

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

中图分类号:

R318.08

梁国新, 梁茵茹, 刘淑贤, 陈康振, 林勇, 秦江霞, 吴洪福. PF-127-LV-NTFs三维复合支架培养大鼠神经干细胞[J]. 基础医学与临床, 2022, 42(1): 75-81.

LIANG Guo-xin, LIANG Yin-ru, LIU Shu-xian, CHEN Kang-zhen, LIN Yong, QIN Jiang-xia, WU Hong-fu. PF-127-LV-NTFs three-dimensional composite scaffold in culturing rat neural stem cells[J]. Basic & Clinical Medicine, 2022, 42(1): 75-81.

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PF-127-LV-NTFs三维复合支架培养大鼠神经干细胞

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

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