碱性成纤维细胞因子对SD大鼠表皮干细胞分化为神经干细胞的影响
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摘要
目的研究碱性成纤维细胞因子(bFGF)对大鼠表皮干细胞分化为神经细胞的影响。方法分离获得饲养了1～3天的新生SD大鼠表皮基底层组织,利用10 min快速贴壁法消化、分离获得表皮干细胞,于倒置显微镜下观察表皮干细胞的形态。培养表皮干细胞的培养基为K-SFM,然后按照不同密度表皮干细胞进行分组处理(0.1×10~7/mL,0.3×10~7/mL,0.5×10~7/mL,0.1×10~6/mL),每组加入20 ng/mL的bFGF,利用细胞免疫组织化学法检测细胞标志物Nestin和NSE的变化,以及观察细胞形态发生的变化。结果成功分离得到SD大鼠表皮干细胞;bFGF诱导后,0.3×10~7/mL组和0.1×10~7/mL组细胞第3天即可见细胞开始伸展生长,在约1周时细胞开始分化成神经细胞;且在细胞形态发生双极化改变的数目趋势上也具有一致性;Nestin和NSE检测均呈阳性表达。结论 bFGF有助于诱导表皮干细胞向神经细胞的分化,而且该诱导作用具有一定的细胞密度依赖性。
Objective To investigate the effect of basic fibroblast factor(bFGF) on the differentiation of epidermal stem cells into nerve cells in rats.Methods The epidermal basal layer tissue of newborn SD rats(1-3 day) were isolated and obtained.Epidermal stem cells were digested and isolatedby rapid attachment to a substrate,and the morphology of epidermal stem cells were observed under an inverted microscope.Epidermal stem cells were cultured with K-SFM culture medium.The epidermal stem cells were grouped according to the density as 0.1×10~7/mL,0.3×10~7/mL,0.5×10~7/mL,0.1×10~6/mL,and each group was added bFGF(20 ng/mL).The changes of cell markers Nestin and NSE were detected by immunohistochemistry,and the changes of cell morphology were observed.Results The epidermal stem cells of SD rat were isolated successfully.After bFGF induction,the 0.3×10~7/mL and 0.1×10~7/mL groups began to stretch growth in the third days,and the cells began to differentiate into nerve cells in about a week.The trend of the number of double polarization changes in the cell morphology was also consistent Nestin and NSE was positively expressed in those cells.Conclusion bFGF was helpful to induce the differentiation of epidermal stem cells into nerve cells,and the induction effect was dependent on the cell density.
Objective To investigate the effect of basic fibroblast factor(bFGF) on the differentiation of epidermal stem cells into nerve cells in rats.Methods The epidermal basal layer tissue of newborn SD rats(1-3 day) were isolated and obtained.Epidermal stem cells were digested and isolatedby rapid attachment to a substrate,and the morphology of epidermal stem cells were observed under an inverted microscope.Epidermal stem cells were cultured with K-SFM culture medium.The epidermal stem cells were grouped according to the density as 0.1×10~7/mL,0.3×10~7/mL,0.5×10~7/mL,0.1×10~6/mL,and each group was added bFGF(20 ng/mL).The changes of cell markers Nestin and NSE were detected by immunohistochemistry,and the changes of cell morphology were observed.Results The epidermal stem cells of SD rat were isolated successfully.After bFGF induction,the 0.3×10~7/mL and 0.1×10~7/mL groups began to stretch growth in the third days,and the cells began to differentiate into nerve cells in about a week.The trend of the number of double polarization changes in the cell morphology was also consistent Nestin and NSE was positively expressed in those cells.Conclusion bFGF was helpful to induce the differentiation of epidermal stem cells into nerve cells,and the induction effect was dependent on the cell density.
引文
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[12] 吕淑坤.表皮干细胞在组织工程创面修复中的应用[J].中国组织工程研究,2012,16(27):5126-5130.
[13] 代学良,程峰,郝怀勇,等.皮肤源性再程序化神经干细胞的制备以及定向分化为神经细胞的体外研究[J].南京医科大学学报(自然科学版),2011,31(3):319-323.
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[15] MALKOWSKI A,SOBOLEWSKI K,JAWORSKI S,et al.FGF binding by extracellular matrix components of wharton′s jelly[J].Acta Biochimica Polonica,2007,54(2):357-363.
[16] 孙钦策,田卫东.碱性成纤维细胞生长因子研究进展[J].现代生物医学进展,2009,9(15):2947-2949.
[17] 汪皖,杨文宇,陈志方.成纤维细胞生长因子在脱位牙再植术中应用的临床研究[J].安徽医药,2015,19(12):2401-2403.
[18] WANG X,LIN G,MARTINSTAYLOR K,et al.Inhibition of caspase-mediated anoikis is critical for basic fibroblast growth factor-sustained culture of human pluripotent stem cells [J].Journal of Biological Chemistry,2009,284(49):34054-34064.
[19] LEE JG,KAY EP.PI 3-kinase/Rac1 and ERK1/2 regulate FGF-2-mediated cell proliferation through phosphorylation of p27 at Ser10 by KIS and at Thr187 by Cdc25A/Cdk2 [J].Investigative Ophthalmology & Visual Science,2011,52(1):417-426.
[20] YANG F,LIU Y,TU J,et al.Activated astrocytes enhance the dopaminergic differentiation of stem cells and promote brain repair through bFGF [J].Nature Communications,2014,5:5627.
[21] DUOBLES T,LIMA TS,LEVY BF,et al.S100beta and fibroblast growth factor-2 are present in cultured Schwann cells and may exert paracrine actions on the peripheral nerve injury [J].Acta Cirurgica Brasileira,2008,23(6):555-560.
[22] 刘晓刚,邓宇斌,蔡辉.碱性成纤维细胞生长因子与猴骨髓间充质干细胞增殖及向神经元前体细胞分化:不同质量浓度对诱导剂隐丹参酮作用有差异吗?[J].中国组织工程研究与临床康复,2010,14(10):1813-1816.
[23] 陈亚男.红景天苷通过ERK1/2和PI_3K/AKT/mTOR信号通路介导小鼠骨髓间充质干细胞向神经元样细胞定向分化的研究[D].兰州:兰州大学,2014.
[24] 张俊虎,王建交,郑永日,等.神经干细胞的鉴定[J].中国组织工程研究,2012,16(27):5108-5112.
[2] 赵宇,孙立伟,姜锐,等.碱性成纤维细胞生长因子修复功能及其应用研究进展[J].中国老年学杂志,2012,32(11):2418-2419.
[3] XIANG Q,XIAO J,ZHANG H,et al.Preparation and characterization of bFGF-encapsulated liposomes and evaluation of wound-healing activities in the rat [J].Burns,2011,37(5):886-895.
[4] CHOI JW,KIM S,KIM TM,et al.Basic fibroblast growth factor activates MEK/ERK cell signaling pathway and stimulates the proliferation of chicken primordial germ cells [J].Plos One,2010,5(9):e12968.DOI:1.1371/journal.pone.0012968.
[5] 陈佳,罗成群.碱性成纤维细胞生长因子(bFGF)与创伤愈合[J].中国烧伤创疡杂志,2005,17(1):74-76.
[6] 佟雷,季丽莉,解大龙,等.碱性成纤维生长因子和神经生长因子联合应用培养成年大鼠海马神经干细胞向神经元样细胞的分化[J].中国组织工程研究,2011,15(1):74-77.
[7] 窦婷婷,满孝勇,郑敏.皮肤干细胞的研究进展[C].2013浙江省医学会皮肤病学学术年会论文汇编,2013.
[8] 杨丽,苏宁,李新实,等.人皮肤表皮干细胞体外培养与应用研究进展[J].现代生物医学进展,2016,16(21):4185-4189,4200.
[9] 刘雪婷,白春雨,关伟军,等.表皮干细胞的生物学特性及其潜在应用[J].生物技术通报,2016(1):29-32.
[10] 王菁,李毅明,程波.快速贴壁法分离小鼠皮肤干细胞的研究[J].皮肤病与性病,2013,35(4):187-188.
[11] SCHEPELER T,PAGE ME,JENSEN KB.Heterogeneity and plasticity of epidermal stem cells [J].Development,2014,141(13):2559-2567.
[12] 吕淑坤.表皮干细胞在组织工程创面修复中的应用[J].中国组织工程研究,2012,16(27):5126-5130.
[13] 代学良,程峰,郝怀勇,等.皮肤源性再程序化神经干细胞的制备以及定向分化为神经细胞的体外研究[J].南京医科大学学报(自然科学版),2011,31(3):319-323.
[14] HU YF,GOURAB K,WELLS C,et al.Epidermal neural crest stem cell (EPI-NCSC)——mediated recovery of sensory function in a mouse model of spinal cord injury [J].Stem Cell Reviews and Reports,2010,6(2):186-198.
[15] MALKOWSKI A,SOBOLEWSKI K,JAWORSKI S,et al.FGF binding by extracellular matrix components of wharton′s jelly[J].Acta Biochimica Polonica,2007,54(2):357-363.
[16] 孙钦策,田卫东.碱性成纤维细胞生长因子研究进展[J].现代生物医学进展,2009,9(15):2947-2949.
[17] 汪皖,杨文宇,陈志方.成纤维细胞生长因子在脱位牙再植术中应用的临床研究[J].安徽医药,2015,19(12):2401-2403.
[18] WANG X,LIN G,MARTINSTAYLOR K,et al.Inhibition of caspase-mediated anoikis is critical for basic fibroblast growth factor-sustained culture of human pluripotent stem cells [J].Journal of Biological Chemistry,2009,284(49):34054-34064.
[19] LEE JG,KAY EP.PI 3-kinase/Rac1 and ERK1/2 regulate FGF-2-mediated cell proliferation through phosphorylation of p27 at Ser10 by KIS and at Thr187 by Cdc25A/Cdk2 [J].Investigative Ophthalmology & Visual Science,2011,52(1):417-426.
[20] YANG F,LIU Y,TU J,et al.Activated astrocytes enhance the dopaminergic differentiation of stem cells and promote brain repair through bFGF [J].Nature Communications,2014,5:5627.
[21] DUOBLES T,LIMA TS,LEVY BF,et al.S100beta and fibroblast growth factor-2 are present in cultured Schwann cells and may exert paracrine actions on the peripheral nerve injury [J].Acta Cirurgica Brasileira,2008,23(6):555-560.
[22] 刘晓刚,邓宇斌,蔡辉.碱性成纤维细胞生长因子与猴骨髓间充质干细胞增殖及向神经元前体细胞分化:不同质量浓度对诱导剂隐丹参酮作用有差异吗?[J].中国组织工程研究与临床康复,2010,14(10):1813-1816.
[23] 陈亚男.红景天苷通过ERK1/2和PI_3K/AKT/mTOR信号通路介导小鼠骨髓间充质干细胞向神经元样细胞定向分化的研究[D].兰州:兰州大学,2014.
[24] 张俊虎,王建交,郑永日,等.神经干细胞的鉴定[J].中国组织工程研究,2012,16(27):5108-5112.