b-FGF壳聚糖载体缓释对神经干细胞分化为神经元的作用
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摘要
目的探讨b-FGF壳聚糖载体(Chitosan carrier)缓释对神经干细胞分化为神经元的作用。方法原代培养和鉴定神经干细胞,随机分为b-FGF壳聚糖缓释组,壳聚糖对照组和b-FGF对照组,Image-pro Plus 2. 0图像分析软件测量细胞突起长度,免疫组织化学方法检测分化细胞中NSE阳性细胞比例,Western blot法检测分化细胞中βⅢ-tubulin的表达和Akt磷酸化程度。结果 48h诱导后,诱导壳聚糖对照组、b-FGF对照组和b-FGF壳聚糖缓释组神经元的分化率分别为38. 5%、42. 6%和70. 8%。b-FGF壳聚糖缓释组与壳聚糖对照组神经元分化率上升(P <0. 05),b-FGF壳聚糖缓释组与b-FGF对照组相比神经元分化率显著提高(P <0. 05);诱导48h后,b-FGF壳聚糖缓释组与壳聚糖对照组和b-FGF对照组相比,神经元突起长度均出现显著增长,差异有统计学意义(P <0. 05); b-FGF壳聚糖缓释组与壳聚糖对照组和b-FGF对照组相比βⅢ-tubulin表达显著增加(P <0.05);诱导5、15和30min后,与b-FGF对照组相比,b-FGF壳聚糖缓释组Akt磷酸化水平显著升高,差异有统计学意义(P <0. 05)。结论 b-FGF经壳聚糖载体缓释后诱导神经元突起形成,提高神经元分化率,Akt磷酸化激活的信号途径可能参与b-FGF壳聚糖载体诱导神经干细胞过程神经元分化速度的调节。
Objective To investigate the effects of b-FGF on the differentiation and formation of neurons from neural stem cells were investigated by Chitosan carrier. Methods The cultured neural stem cells by primary sources,the experiment was divided into b-FGF sustained-releasing chitosan group,chitosan control group and b-FGF control group. Affect the measurement of Chitosan differentiation group and b-FGF differentiation group and Chitosan sustained-releasing groups of the neurite formation,using image analysis software Image-pro Plus2. 0 image analysis software to measure cell neurite length in data acquisition. Immunohistochemical method was to check cell differentiation the ratio of NSE positive cells. Western blot method was performed to detect the expression of Akt phosphorylation. Results The induction of differentiation of neural stem cells into neuronal differentiation rate were 38. 5%(Chitosan group),42. 6%( b-FGF group)and 70. 8%(b-FGF Chitosan sustained-releasing group),the differentiation rate of neurons in b-FGF chitosan sustained-releasing group was higher than that in the chitosan control group(P < 0. 05),and the differentiation rate of neurons in b-FGF chitosan sustained-releasing group was higher than that in b-FGF control group(P < 0. 05);48 hours after induction,the neurite length of the b-FGF chitosan sustained-releasing group increased compared with that in the chitosan control group and the b-FGF control group,and the difference was statistically significant(P < 0. 05);The expression of βⅢ-tubulin was increased in b-FGF chitosan sustainedreleasing group compared with chitosan control group and b-FGF control group(P < 0. 05);After 5 minutes,15 minutes and 30 minutes of induction,the phosphorylation level of Akt in b-FGF chitosan sustainedreleasing group was higher than that in b-FGF control group,and the difference was statistically significant(P< 0. 05). Conclusion B-FGF could induce neurite formation and increase the differentiation rate of neurons after sustained release by chitosan carrier. Akt phosphorylation-activated signaling pathway may be involved in the regulation of neuronal differentiation rate in the process of b-FGF chitosan vector-induced neural stem cells.
Objective To investigate the effects of b-FGF on the differentiation and formation of neurons from neural stem cells were investigated by Chitosan carrier. Methods The cultured neural stem cells by primary sources,the experiment was divided into b-FGF sustained-releasing chitosan group,chitosan control group and b-FGF control group. Affect the measurement of Chitosan differentiation group and b-FGF differentiation group and Chitosan sustained-releasing groups of the neurite formation,using image analysis software Image-pro Plus2. 0 image analysis software to measure cell neurite length in data acquisition. Immunohistochemical method was to check cell differentiation the ratio of NSE positive cells. Western blot method was performed to detect the expression of Akt phosphorylation. Results The induction of differentiation of neural stem cells into neuronal differentiation rate were 38. 5%(Chitosan group),42. 6%( b-FGF group)and 70. 8%(b-FGF Chitosan sustained-releasing group),the differentiation rate of neurons in b-FGF chitosan sustained-releasing group was higher than that in the chitosan control group(P < 0. 05),and the differentiation rate of neurons in b-FGF chitosan sustained-releasing group was higher than that in b-FGF control group(P < 0. 05);48 hours after induction,the neurite length of the b-FGF chitosan sustained-releasing group increased compared with that in the chitosan control group and the b-FGF control group,and the difference was statistically significant(P < 0. 05);The expression of βⅢ-tubulin was increased in b-FGF chitosan sustainedreleasing group compared with chitosan control group and b-FGF control group(P < 0. 05);After 5 minutes,15 minutes and 30 minutes of induction,the phosphorylation level of Akt in b-FGF chitosan sustainedreleasing group was higher than that in b-FGF control group,and the difference was statistically significant(P< 0. 05). Conclusion B-FGF could induce neurite formation and increase the differentiation rate of neurons after sustained release by chitosan carrier. Akt phosphorylation-activated signaling pathway may be involved in the regulation of neuronal differentiation rate in the process of b-FGF chitosan vector-induced neural stem cells.
引文
[1]Bramlett HM,Dietrich WD.Long-term consequences of traumatic brain injury:current status of potential mechanisms of injury and neurological outcomes[J].J Neurotrauma,2015,32(23):1834-1848.DOI:10.1089/neu.2014.3352.
[2]Mahmoud AA,Salama AH.Norfloxacin-loaded collagen/chitosan scaffolds for skin reconstruction:preparation,evaluation and in-vivo wound healing assessment[J].Eur J Pharm Sci,2016,83:155-165.DOI:10.1016/j.ejps.2015.12.026.
[3]Yamamoto K,Tajima Y,Hasegawa A,et al.Contrasting effects of stanniocalcin-related polypeptides on macrophage foam cell formation and vascular smooth muscle cell migration[J].Peptides,2016,82:120-127.DOI:10.1016/j.peptides.2016.06.009.
[4]王光辉,钟鸣,张敏娜.丹参注射液体外对神经干细胞分化细胞突起形成的影响[J].中风与神经疾病杂志,2018,35(7):580-584.
[5]Yang Z,Zhang A,Duan H,et al.NT3-chitosan elicits robust endogenous neurogenesis to enable functional recovery after spinal cord injury[J].Proc Natl Acad Sci USA,2015,112(43):13354-13359.DOI:10.1073/pnas.1510194112.
[6]Duan H,Ge W,Zhang A,et al.Transcriptome analyses reveal molecular mechanisms underlying functional recovery after spinal cord injury[J].Proc Natl Acad Sci USA,2015,112(43):13360-13365.DOI:10.1073/pnas.1510176112.
[7]Palmer TD,Markakis EA,Willhoite AR,et al.Fibroblast growth factor-2 activates a latent neurogenic program in neural stem cells from diverse regions of the adult CNS[J].J Neurosci,1999,19(19):8487-8497.DOI:10.1523/jneurosci.19-19-08487.1999.
[8]翟婧妍,段红梅,尚俊奎,等.碱性成纤维细胞生长因子-壳聚糖载体诱导神经干细胞分化及与肌细胞的共培养[J].中国组织工程研究,2017,21(6):877-882.
[9]周靖,包丽雯,梁静.神经干细胞移植帕金森病模型大鼠行为学及对炎症因子的调节效应[J].中国组织工程研究,2017,21(33):5299-5304.
[10]许刚,赵晨光,孙玮,等.LINGO-1在脊髓神经干细胞分化过程中的表达特征及效应[J].中国组织工程研究,2017,21(33):5394-5399.
[11]王光辉,钟鸣,郑公朴,等.青蒿素对老年小鼠学习记忆及炎性细胞因子和单胺类神经递质的影响[J].中华行为医学与脑科学杂志,2018,27(7):593-597.DOI:10.3760/cma.j.issn.1674-6554.2018.07.004
[12]Tanaka Y,Isomura T,Shimba K,et al.Neurogenesis enhances response specificity to spatial pattern stimulation in hippocampal cultures[J].IEEE Trans Biomed Eng,2017,64(11):2555-2561.DOI:10.1109/TBME.2016.2639468.
[13]Ladeira K,Macedo F,Longatto-Filho A,et al.Angiogenic factors:role in esophageal cancer,a brief review[J].Esophagus,2018,15(2):53-58.DOI:10.1007/s10388-017-0597-1.
[14]Zhang JJ,Zhu JJ,Hu YB,et al.Transplantation of bFGFexpressing neural stem cells promotes cell migration and functional recovery in rat brain after transient ischemic stroke[J].Oncotarget,2017,8(60):102067-102077.DOI:10.18632/oncotarget.22155.
[15]Li C,Che LH,Shi L,et al.Suppression of basic fibroblast growth factor expression by antisense oligonucleotides inhibits neural stem cell proliferation and differentiation in rat models with focal cerebral infarction[J].JCell Biochem,2017,118(11):3875-3882.DOI:10.1002/jcb.26038.
[16]Abouzaripour M,Fathi F,Daneshi E,et al.Combined effect of retinoic acid and basic fibroblast growth factor on maturation of mouse oocyte and subsequent fertilization and development[J].Int J Fertil Steril,2018,12(1):68-71.DOI:10.22074/ijfs.2018.5293.
[17]Wei Z,Zhao J,Chen YM,et al.Self-healing polysaccharidebased hydrogels as injectable carriers for neural stem cells[J].Sci Rep,2016,6:37841.DOI:10.1038/srep37841.
[18]Moore L,Skop NB,Rothbard DE,et al.Tethered growth factors on biocompatible scaffolds improve stemness of cultured rat and human neural stem cells and growth of oligodendrocyte progenitors[J].Methods,2018,133:54-64.DOI:10.1016/j.ymeth.2017.08.015.
[2]Mahmoud AA,Salama AH.Norfloxacin-loaded collagen/chitosan scaffolds for skin reconstruction:preparation,evaluation and in-vivo wound healing assessment[J].Eur J Pharm Sci,2016,83:155-165.DOI:10.1016/j.ejps.2015.12.026.
[3]Yamamoto K,Tajima Y,Hasegawa A,et al.Contrasting effects of stanniocalcin-related polypeptides on macrophage foam cell formation and vascular smooth muscle cell migration[J].Peptides,2016,82:120-127.DOI:10.1016/j.peptides.2016.06.009.
[4]王光辉,钟鸣,张敏娜.丹参注射液体外对神经干细胞分化细胞突起形成的影响[J].中风与神经疾病杂志,2018,35(7):580-584.
[5]Yang Z,Zhang A,Duan H,et al.NT3-chitosan elicits robust endogenous neurogenesis to enable functional recovery after spinal cord injury[J].Proc Natl Acad Sci USA,2015,112(43):13354-13359.DOI:10.1073/pnas.1510194112.
[6]Duan H,Ge W,Zhang A,et al.Transcriptome analyses reveal molecular mechanisms underlying functional recovery after spinal cord injury[J].Proc Natl Acad Sci USA,2015,112(43):13360-13365.DOI:10.1073/pnas.1510176112.
[7]Palmer TD,Markakis EA,Willhoite AR,et al.Fibroblast growth factor-2 activates a latent neurogenic program in neural stem cells from diverse regions of the adult CNS[J].J Neurosci,1999,19(19):8487-8497.DOI:10.1523/jneurosci.19-19-08487.1999.
[8]翟婧妍,段红梅,尚俊奎,等.碱性成纤维细胞生长因子-壳聚糖载体诱导神经干细胞分化及与肌细胞的共培养[J].中国组织工程研究,2017,21(6):877-882.
[9]周靖,包丽雯,梁静.神经干细胞移植帕金森病模型大鼠行为学及对炎症因子的调节效应[J].中国组织工程研究,2017,21(33):5299-5304.
[10]许刚,赵晨光,孙玮,等.LINGO-1在脊髓神经干细胞分化过程中的表达特征及效应[J].中国组织工程研究,2017,21(33):5394-5399.
[11]王光辉,钟鸣,郑公朴,等.青蒿素对老年小鼠学习记忆及炎性细胞因子和单胺类神经递质的影响[J].中华行为医学与脑科学杂志,2018,27(7):593-597.DOI:10.3760/cma.j.issn.1674-6554.2018.07.004
[12]Tanaka Y,Isomura T,Shimba K,et al.Neurogenesis enhances response specificity to spatial pattern stimulation in hippocampal cultures[J].IEEE Trans Biomed Eng,2017,64(11):2555-2561.DOI:10.1109/TBME.2016.2639468.
[13]Ladeira K,Macedo F,Longatto-Filho A,et al.Angiogenic factors:role in esophageal cancer,a brief review[J].Esophagus,2018,15(2):53-58.DOI:10.1007/s10388-017-0597-1.
[14]Zhang JJ,Zhu JJ,Hu YB,et al.Transplantation of bFGFexpressing neural stem cells promotes cell migration and functional recovery in rat brain after transient ischemic stroke[J].Oncotarget,2017,8(60):102067-102077.DOI:10.18632/oncotarget.22155.
[15]Li C,Che LH,Shi L,et al.Suppression of basic fibroblast growth factor expression by antisense oligonucleotides inhibits neural stem cell proliferation and differentiation in rat models with focal cerebral infarction[J].JCell Biochem,2017,118(11):3875-3882.DOI:10.1002/jcb.26038.
[16]Abouzaripour M,Fathi F,Daneshi E,et al.Combined effect of retinoic acid and basic fibroblast growth factor on maturation of mouse oocyte and subsequent fertilization and development[J].Int J Fertil Steril,2018,12(1):68-71.DOI:10.22074/ijfs.2018.5293.
[17]Wei Z,Zhao J,Chen YM,et al.Self-healing polysaccharidebased hydrogels as injectable carriers for neural stem cells[J].Sci Rep,2016,6:37841.DOI:10.1038/srep37841.
[18]Moore L,Skop NB,Rothbard DE,et al.Tethered growth factors on biocompatible scaffolds improve stemness of cultured rat and human neural stem cells and growth of oligodendrocyte progenitors[J].Methods,2018,133:54-64.DOI:10.1016/j.ymeth.2017.08.015.