慢病毒介导神经生长因子过表达诱导脐带间充质干细胞向神经元样分化
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摘要
目的探讨神经生长因子(NGF)过表达慢病毒转染脐带间充质干细胞(UMSCs)对细胞分化的影响。方法分离培养UMSCs,流式细胞术鉴定后利用慢病毒载体感染细胞,使其过表达NGF,72h后,通过免疫荧光检测NGF和GFP的表达,Western blot检测NGF蛋白表达水平,ELISA法检测细胞上清液NGF的含量,qRT-PCR检测相关神经因子基因的表达。结果流式细胞术显示细胞表面CD105、CD90、CD73阳性而CD34、CD45、CD19和CD14阴性,鉴定为脐带间充质干细胞。慢病毒转染后细胞NGF和GFP阳性,细胞内NGF和培养液中b-NGF含量都显著性增加,NGF、nestin、GFAP、MAP2及tubulin等mRNA的表达显著性增加。结论神经生长因子过表达慢病毒转染脐带间充质干细胞会促使细胞向神经元样细胞分化,可应用于后续研究。
Objective To investigate the effect of nerve growth factor(NGF) overexpression via lentivirus transfection on the differentiation of umbilical cord mesenchymal stem cells(UMSCs). Methods UMSCs were isolated and cultured, then identified by flow cytometry, and then transfected with lentiviral vectors to overexpress NGF. After 72 hours, the expression of NGF and GFP was detected by immunofluorescence, and the expression of NGF protein was detected by Western blot. In the cell supernatant, the content of NGF was detected by ELISA and the expression of related neural factor genes was detected by qRT-PCR. Results Flow cytometry results showed that cells were CD90, CD73 and CD105 positive, CD34, CD45, CD19 and CD14 negative on the surface, which identified the cells as UMSCs. After lentivirus transfection, it was found that the cells were positive for NGF and GFP, both content of intracellular NGF and supernatant NGF increased significantly, furthermore mRNA expression of NGF, nestin, GFAP, MAP2, and tubulin was increased significantly. Conclusion Nerve growth factor overexpression via lentiviral transfection of umbilical cord mesenchymal stem cells promotes differentiation of cells into neuron-like cells, which can be used in subsequent studies.
Objective To investigate the effect of nerve growth factor(NGF) overexpression via lentivirus transfection on the differentiation of umbilical cord mesenchymal stem cells(UMSCs). Methods UMSCs were isolated and cultured, then identified by flow cytometry, and then transfected with lentiviral vectors to overexpress NGF. After 72 hours, the expression of NGF and GFP was detected by immunofluorescence, and the expression of NGF protein was detected by Western blot. In the cell supernatant, the content of NGF was detected by ELISA and the expression of related neural factor genes was detected by qRT-PCR. Results Flow cytometry results showed that cells were CD90, CD73 and CD105 positive, CD34, CD45, CD19 and CD14 negative on the surface, which identified the cells as UMSCs. After lentivirus transfection, it was found that the cells were positive for NGF and GFP, both content of intracellular NGF and supernatant NGF increased significantly, furthermore mRNA expression of NGF, nestin, GFAP, MAP2, and tubulin was increased significantly. Conclusion Nerve growth factor overexpression via lentiviral transfection of umbilical cord mesenchymal stem cells promotes differentiation of cells into neuron-like cells, which can be used in subsequent studies.
引文
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[9]Gates MA, Fricker-Gates RA, Macklis JD. Reconstruction of cortical circuitry. Prog Brain Res, 2000, 127(127):115-156.
[10] Vawda R, Fehlings MG. Mesenchymal cells in the treatment of spinal cord injury:current&future perspectives. Curr Stem Cell Res Ther, 2013, 8(1):25-38.
[11] Liu J, Han D, Wang Z, et al. Clinical analysis of the treatment of spinal cord injury with umbilical cord mesenchymal stem cells. Cytotherapy, 2011, 15(2):185-191.
[12] Xiong N, Zhang Z, Huang J, et al. VEGF-expressing human umbilical cord mesenchymal stem cells, an improved therapy strategy for Parkinson’s disease. Gene Ther, 2011, 18(4):394-402.
[13] Shih YY, Nakagawara A, Lee H, et al. Calreticulinmediates nerve growth factor-induced neuronal differentiation. J Mol Neurosci, 2012, 47(3):571.
[14] Feng Z, Li K, Mei L, et al. NRAGE is a negative regulator of nerve growth factor-stimulated neurite outgrowth in PC12cellsmediatedthrough TrkA-ERKsignaling. JNeurosci Res, 2010, 88(8):1822–1828.
[15] Park D, Xiang AP, Mao FF, et al. Nestin is required for the proper self-renewal of neural stem cells. Stem Cells, 2010,28(12):2162.
[16] Mortazavi Y, Sheikhsaran F, Soleimani M, et al. The evaluation of nerve growth factor over expressionon neural lineage specific genes in human mesenchymal stem cells. Cell J,2016, 18(2):189-196.
[17]陈俊,杨自金.鼠神经生长因子体外诱导脐血间充质干细胞向类神经元分化.中国组织工程研究,2018,22(13):2027-2032.
[2]Pittenger MF, Mackay AM, Beck SC, et al. Multilineage Potential of Adult Human Mesenchymal Stem Cells. Science,1999, 284(5411):143-147.
[3]XuR,JiangX,GuoZ,etal.Functionalanalysisofneuron-like cells differentiated from neural stem cells derived from bone marrow stroma cells in vitro. Cell Mol Neurobiol,2008, 28(4):545-558.
[4]Caplan AI. Adult mesenchymal stem cells for tissue engineering versus regenerative medicine. J Cell Physiol, 2007,213(2):341–347.
[5]Tischler AS, Riseberg JC, Hardenbrook MA, et al. Nerve growth factor is a potent inducer of proliferation and neuronal differentiation for adult rat chromaffin cells in vitro. J Neurosci, 1993, 13(4):1533-1542.
[6]Lewin GR, Ritter AM, Mendell LM. Nerve growth factor-induced hyperalgesia in the neonatal and adult rat. J Neurosci,1993, 13(5):2136-2148.
[7]Sumbria RK, Boado RJ, Pardridge WM. Combination stroke therapy in the mouse with blood-brain barrier penetrating IgG-GDNF and IgG-TNF decoy receptor fusion proteins.Brain Res, 2013, 1507(17):91-96.
[8]Bj?rklund A,LindvallO.Cellreplacementtherapiesfor central nervous system disorders. Nat Neurosci, 2000, 3(6):537-544.
[9]Gates MA, Fricker-Gates RA, Macklis JD. Reconstruction of cortical circuitry. Prog Brain Res, 2000, 127(127):115-156.
[10] Vawda R, Fehlings MG. Mesenchymal cells in the treatment of spinal cord injury:current&future perspectives. Curr Stem Cell Res Ther, 2013, 8(1):25-38.
[11] Liu J, Han D, Wang Z, et al. Clinical analysis of the treatment of spinal cord injury with umbilical cord mesenchymal stem cells. Cytotherapy, 2011, 15(2):185-191.
[12] Xiong N, Zhang Z, Huang J, et al. VEGF-expressing human umbilical cord mesenchymal stem cells, an improved therapy strategy for Parkinson’s disease. Gene Ther, 2011, 18(4):394-402.
[13] Shih YY, Nakagawara A, Lee H, et al. Calreticulinmediates nerve growth factor-induced neuronal differentiation. J Mol Neurosci, 2012, 47(3):571.
[14] Feng Z, Li K, Mei L, et al. NRAGE is a negative regulator of nerve growth factor-stimulated neurite outgrowth in PC12cellsmediatedthrough TrkA-ERKsignaling. JNeurosci Res, 2010, 88(8):1822–1828.
[15] Park D, Xiang AP, Mao FF, et al. Nestin is required for the proper self-renewal of neural stem cells. Stem Cells, 2010,28(12):2162.
[16] Mortazavi Y, Sheikhsaran F, Soleimani M, et al. The evaluation of nerve growth factor over expressionon neural lineage specific genes in human mesenchymal stem cells. Cell J,2016, 18(2):189-196.
[17]陈俊,杨自金.鼠神经生长因子体外诱导脐血间充质干细胞向类神经元分化.中国组织工程研究,2018,22(13):2027-2032.