3D共培养模型对体外培养兔角膜缘干细胞的影响
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摘要
目的探讨三维(three-dimension,3D)共培养模型对体外扩增兔角膜缘干细胞(limbal stem cells,LSCs)的作用。方法体外培养兔LSCs,以牙周膜干细胞(human periodontal stem cell,HPDLSCs)作为饲细胞建立共培养模型,根据载体不同分为3组,纤维蛋白胶组:将兔LSCs、HPDLSCs、纤维蛋白胶3D共培养;羊膜组:将兔LSCs、HPDLSCs、羊膜3D共培养;对照组:将兔LSCs、HPDLSCs共培养。共培养5 d后,用倒置显微镜、HE染色、扫描电镜观察兔LSCs的细胞形态,采用免疫荧光染色检测各组兔LSCs阳性标志物p63的表达,并比较各组p63的阳性率。结果共培养5 d后,倒置显微镜、HE染色观察细胞形态,羊膜组、纤维蛋白胶组细胞排列紧密,呈大小不等的团块状集落,细胞大小基本一致;对照组细胞排列相对稀疏;扫描电镜下观察见纤维蛋白胶组和羊膜组细胞与载体贴附良好。免疫荧光染色显示纤维蛋白胶组、羊膜组和对照组LSCs的p63阳性率分别为(69.93±8.76)%、(78.36±8.56)%和(58.59±8.31)%,三组间总体比较差异有统计学意义(F=9.43,P=0.000)。两两比较中纤维蛋白胶组、羊膜组LSCs的p63阳性率均大于对照组,差异均有统计学意义(均为P<0.05)。纤维蛋白胶组、羊膜组的p63阳性率比较,差异无统计学意义(P>0.05)。结论以纤维蛋白胶、羊膜作为载体,HPDLSCs作为饲细胞的3D共培养模型有助于体外扩增兔LSCs。
Objective To investigate the effect of three-dimensional(3 D) co-culture model on the expansion of rabbit limbal stem cells in vitro.Methods The rabbit limbal stem cells(LSCs) were cultured in vitro,and the human periodontal stem cells(HPDLSCs) were served as the feeder cells to establish of co-culture model.According to different carriers,the cells were divided into three groups:the fibrin glue group,in which LSCs were co-cultured with fibrin glue and HPDLSCs;the amniotic membrane group,in which LSCs were co-cultured with amniotic membrane and HPDLSCs;and the control group,in which LSCs were co-cultured only with the HPDLSCs.After co-culture for 5 days,we used inverted microscope,hematoxylin and eosin staining and scanning electron microscopy to observe the cells morphology,and then used the immunofluorescence staining to detect the expression of the LSCs positive marker p63,followed by the comparison of the positive rate of p63 in each group.Results After co-cultured for five days,the results from inverted microscope and hematoxylin and eosin staining showed that cells in the fibrin glue group and the amniotic membrane group were uniform and serried arrangement,and presented cluster-like colonies with varying sizes,and cell sizes were uniformly,whereas cells in the control group were relatively sparse.Scanning electron microscopy showed that the cells were well adhered to the carrier in the fibrin glue group and the amniotic membrane group.Immunofluorescence staining showed that the positive rates of p63 in the fibrin glue group,the amniotic membrane group,the control group were(69.93±8.76)%,(78.36±8.56)%,(58.59±8.31)%,respectively,and there was significant difference among the three groups(F=9.43,P=0.000).In pairwise comparison,the positive rates of p63 in the fibrin glue group and amniotic membrane group were both higher than those of the control group,and there was significant differences(both P<0.05),but there was no significant difference in the positive rate of p63 between the fibrin glue group and amniotic membrane group(P>0.05).Conclusion The co-culture 3 D model is helpful for in vivo expansion of rabbit LSCs.
Objective To investigate the effect of three-dimensional(3 D) co-culture model on the expansion of rabbit limbal stem cells in vitro.Methods The rabbit limbal stem cells(LSCs) were cultured in vitro,and the human periodontal stem cells(HPDLSCs) were served as the feeder cells to establish of co-culture model.According to different carriers,the cells were divided into three groups:the fibrin glue group,in which LSCs were co-cultured with fibrin glue and HPDLSCs;the amniotic membrane group,in which LSCs were co-cultured with amniotic membrane and HPDLSCs;and the control group,in which LSCs were co-cultured only with the HPDLSCs.After co-culture for 5 days,we used inverted microscope,hematoxylin and eosin staining and scanning electron microscopy to observe the cells morphology,and then used the immunofluorescence staining to detect the expression of the LSCs positive marker p63,followed by the comparison of the positive rate of p63 in each group.Results After co-cultured for five days,the results from inverted microscope and hematoxylin and eosin staining showed that cells in the fibrin glue group and the amniotic membrane group were uniform and serried arrangement,and presented cluster-like colonies with varying sizes,and cell sizes were uniformly,whereas cells in the control group were relatively sparse.Scanning electron microscopy showed that the cells were well adhered to the carrier in the fibrin glue group and the amniotic membrane group.Immunofluorescence staining showed that the positive rates of p63 in the fibrin glue group,the amniotic membrane group,the control group were(69.93±8.76)%,(78.36±8.56)%,(58.59±8.31)%,respectively,and there was significant difference among the three groups(F=9.43,P=0.000).In pairwise comparison,the positive rates of p63 in the fibrin glue group and amniotic membrane group were both higher than those of the control group,and there was significant differences(both P<0.05),but there was no significant difference in the positive rate of p63 between the fibrin glue group and amniotic membrane group(P>0.05).Conclusion The co-culture 3 D model is helpful for in vivo expansion of rabbit LSCs.
引文
[1] PELLEGRINI G,RAMA P,DI R A,PANARAS A,DE L M.Concise review:hurdles in a successful example of limbal stem cell-based regenerative medicine[J].Stem Cells,2014,32(1):26-34.
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[3] XU B,FAN T J,ZHAO J,SUN A,WANG R X,HU X Z,et al.Transplantation of tissue-engineered human corneal epithelium in limbal stem cell deficiency rabbit models[J].Int J Ophthalmol,2012,5(4):424-429.
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[6] HU R,GAO J,LIANG Y R,HUANG Y,LI H,SU M.An experimental study of cultivating rat limbal stem cells in vitro on amniotic membrane[J].Chongqing Med,2016,45(23):3233-3236.胡蓉,高杰,梁雅茹,黄悦,李红,苏敏.以羊膜为载体体外培养大鼠角膜缘干细胞的实验研究[J].重庆医学,2016,45(23):3233-3236.
[7] ZHOU Y.Effects of feeder layer cell membrane fluidity on induced pluripotent stem cells (miPS) in mice[D].Chang chun:Jilin University,2014.周悦.饲养层细胞膜流动性对小鼠诱导性多能干细胞(miPS)的影响 [D].长春:吉林大学,2014.
[8] WANG H X,GAO X W,CAI Y,LI W J.Effects of periodontal ligament stem cells and human umbilical cord mesenchymal stem cells on microenvironment of corneal limbal stem cells cultured in vitro[J].Rec Adv Ophthal,2016,36(11):1015-1020.王慧娴,高晓唯,蔡岩,李文静.人牙周膜干细胞、脐带间充质干细胞对体外培养角膜缘干细胞的影响[J].眼科新进展,2016,36(11):1015-1020.
[9] ZHANG X,SUN H,LI X,YUAN X,ZHANG L,ZHAO S.Utilization of human limbal mesenchymal cells as feeder layers for human limbal stem cells cultured on amniotic membrane[J].J Tissue Eng Reg Med,2010,4(1):38-44.
[10] MEI H,GONZ LEZ S,NAKATSU M N,BACLAGON E R,CHEN F V,DENG S X.Human adipose-derived stem cells support the growth of limbal stem/progenitor cells[J].PLoS One,2017,12(10):225-240.
[11] ZHANG W,FANG M,BAO D Y,DING X C,TONG X,QIN H Y.Effects of DAPT on the proliferation and osteogenic differentiation of human periodontal ligment stem cells[J].Oral Biomed,2018,9(1):7-11.张文,方明,鲍东昱,丁晓晨,童昕,秦海燕.DAPT对牙周膜干细胞体外增殖及成骨分化的影响[J].口腔生物医学,2018,9(1):7-11.
[12] SAICHANMA S,BUNYARATVEJ A,SILA-ASNA M.In vitro transdifferentiation of corneal epithelial-like cells from human skin-derived precursor cells[J].Int J Ophthalmol,2012,5(2):158-163.
[13] BOURNE G L.The microscopic anatomy of the human amnion and chorion[J].Am J Obstet Gynecol,1960,79(6):1070-1073.
[14] UTHEIM T P,AASS UTHEIM ?,SALVANOS P,JACKSON C J,SCHRADER S,GEERLING G,et al.Concise review:altered versus unaltered amniotic membrane as a substrate for limbal epithelial cells[J].Stem Cells Transl Med,2018,7(5):415-427.
[15] MOHAN R,BAJAJ A,GUNDAPPA M.Human amnion membrane:potential applications in oral and periodontal field[J].J Int Soc Pre Comm Dent,2017,7(1):15-21.
[16] NOORI A,ASHRAFI S J,VAEZ-GHAEMI R,HATAMIAN-ZAREMI A,WEBSTER T J.A review of fibrin and fibrin composites for bone tissue engineering[J].Int J Nanomed,2017,12:4937-4961.
[17] BROWN A C,BARKER T H.Fibrin-based biomaterials:Modulation of macroscopic properties through rational design at the molecular level[J].Acta Biomater,2014,10(4):1502-1514.
[18] LINSLEY C S,WU B M,TAWIL B.Mesenchymal stem cell growth on and mechanical properties of fibrin-based biomimetic bone scaffolds[J].J Biomed Mater Res,2016,104(12):2945-2953.
[19] MEHRZAD B,MILAD M,AZAM S,NAVID N,MANICA N,FATEMEH M,et al.Optimization of three dimensional culturing of the HepG2 cell line in fibrin scaffold[J].Hepat Mon,2015,15(3):e22731.
[20] XIONG L J,ZHENG T,HU Y Z.Co-culture of mouse embryonic fibroblasts and fibrin with corneal limbal stem cells for viability and phenotype maintenance[J].Acta Med Univ Sci Technol Huazhong,2012,41(6):680-683.熊林杰,郑恬,胡义珍.鼠角膜缘干细胞3D共培养模型的建立及生存率、表型维持[J].华中科技大学学报(医学版),2012,41(6):680-683.
[2] SCHL TZER-SCHREHARDT U,KRUSE F E.Identification and characterization of limbal stem cells[J].Exp Eye Res,2005,81(3):247-264.
[3] XU B,FAN T J,ZHAO J,SUN A,WANG R X,HU X Z,et al.Transplantation of tissue-engineered human corneal epithelium in limbal stem cell deficiency rabbit models[J].Int J Ophthalmol,2012,5(4):424-429.
[4] HOSSEINKHANI M,MEHRABANI D,KARIMFAR M H,BAKHTIYARI S,MANAFI A,SHIRAZI R.Tissue engineered scaffolds in regenerative medicine[J].World J Plast Surg,2014,3(1):3-7.
[5] LI Y,GU Z Y,LV Q F,YOU J B,LIU F F.Primary culture of human periodontal ligament stem cells and dental pulp stem cells by tissue enzymatic digestion method[J].J Clin Rehabil Tis Eng Res,2012,16(32):5993-5998.李颖,谷子芽,吕秋峰,尤金彪,刘芳菲.酶解组织块法原代培养人牙周膜干细胞和牙髓干细胞[J].中国组织工程研究,2012,16(32):5993-5998.
[6] HU R,GAO J,LIANG Y R,HUANG Y,LI H,SU M.An experimental study of cultivating rat limbal stem cells in vitro on amniotic membrane[J].Chongqing Med,2016,45(23):3233-3236.胡蓉,高杰,梁雅茹,黄悦,李红,苏敏.以羊膜为载体体外培养大鼠角膜缘干细胞的实验研究[J].重庆医学,2016,45(23):3233-3236.
[7] ZHOU Y.Effects of feeder layer cell membrane fluidity on induced pluripotent stem cells (miPS) in mice[D].Chang chun:Jilin University,2014.周悦.饲养层细胞膜流动性对小鼠诱导性多能干细胞(miPS)的影响 [D].长春:吉林大学,2014.
[8] WANG H X,GAO X W,CAI Y,LI W J.Effects of periodontal ligament stem cells and human umbilical cord mesenchymal stem cells on microenvironment of corneal limbal stem cells cultured in vitro[J].Rec Adv Ophthal,2016,36(11):1015-1020.王慧娴,高晓唯,蔡岩,李文静.人牙周膜干细胞、脐带间充质干细胞对体外培养角膜缘干细胞的影响[J].眼科新进展,2016,36(11):1015-1020.
[9] ZHANG X,SUN H,LI X,YUAN X,ZHANG L,ZHAO S.Utilization of human limbal mesenchymal cells as feeder layers for human limbal stem cells cultured on amniotic membrane[J].J Tissue Eng Reg Med,2010,4(1):38-44.
[10] MEI H,GONZ LEZ S,NAKATSU M N,BACLAGON E R,CHEN F V,DENG S X.Human adipose-derived stem cells support the growth of limbal stem/progenitor cells[J].PLoS One,2017,12(10):225-240.
[11] ZHANG W,FANG M,BAO D Y,DING X C,TONG X,QIN H Y.Effects of DAPT on the proliferation and osteogenic differentiation of human periodontal ligment stem cells[J].Oral Biomed,2018,9(1):7-11.张文,方明,鲍东昱,丁晓晨,童昕,秦海燕.DAPT对牙周膜干细胞体外增殖及成骨分化的影响[J].口腔生物医学,2018,9(1):7-11.
[12] SAICHANMA S,BUNYARATVEJ A,SILA-ASNA M.In vitro transdifferentiation of corneal epithelial-like cells from human skin-derived precursor cells[J].Int J Ophthalmol,2012,5(2):158-163.
[13] BOURNE G L.The microscopic anatomy of the human amnion and chorion[J].Am J Obstet Gynecol,1960,79(6):1070-1073.
[14] UTHEIM T P,AASS UTHEIM ?,SALVANOS P,JACKSON C J,SCHRADER S,GEERLING G,et al.Concise review:altered versus unaltered amniotic membrane as a substrate for limbal epithelial cells[J].Stem Cells Transl Med,2018,7(5):415-427.
[15] MOHAN R,BAJAJ A,GUNDAPPA M.Human amnion membrane:potential applications in oral and periodontal field[J].J Int Soc Pre Comm Dent,2017,7(1):15-21.
[16] NOORI A,ASHRAFI S J,VAEZ-GHAEMI R,HATAMIAN-ZAREMI A,WEBSTER T J.A review of fibrin and fibrin composites for bone tissue engineering[J].Int J Nanomed,2017,12:4937-4961.
[17] BROWN A C,BARKER T H.Fibrin-based biomaterials:Modulation of macroscopic properties through rational design at the molecular level[J].Acta Biomater,2014,10(4):1502-1514.
[18] LINSLEY C S,WU B M,TAWIL B.Mesenchymal stem cell growth on and mechanical properties of fibrin-based biomimetic bone scaffolds[J].J Biomed Mater Res,2016,104(12):2945-2953.
[19] MEHRZAD B,MILAD M,AZAM S,NAVID N,MANICA N,FATEMEH M,et al.Optimization of three dimensional culturing of the HepG2 cell line in fibrin scaffold[J].Hepat Mon,2015,15(3):e22731.
[20] XIONG L J,ZHENG T,HU Y Z.Co-culture of mouse embryonic fibroblasts and fibrin with corneal limbal stem cells for viability and phenotype maintenance[J].Acta Med Univ Sci Technol Huazhong,2012,41(6):680-683.熊林杰,郑恬,胡义珍.鼠角膜缘干细胞3D共培养模型的建立及生存率、表型维持[J].华中科技大学学报(医学版),2012,41(6):680-683.