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。
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.
作者
梁国新
梁茵茹
刘淑贤
陈康振
林勇
秦江霞
吴洪福
LIANG Guo-xin;LIANG Yin-ru;LIU Shu-xian;CHEN Kang-zhen;LIN Yong;QIN Jiang-xia;WU Hong-fu(The Third Affiliated Hospital, Guangdong Medical University (Shunde Longjiang Hospital),Foshan 528318;Guangzhou Huadu Hospital, Guangdong Medical University,Guangzhou 510800;Key Laboratory of Stem Cell and Regenerative Tissue Engineering,Guangdong Medical University, Dongguan 523808, China)
出处
《基础医学与临床》
2022年第1期75-81,共7页
Basic and Clinical Medicine
基金
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)。
