人羊膜上皮细胞培养工艺的优化及生物学特性
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摘要
背景:目前人羊膜上皮细胞分离、培养及冻存的研究较为分散,难以形成全面有效的方案以满足未来对移植用干细胞的临床需求。目的:建立人羊膜上皮细胞优化的分离、培养与冻存工艺,并对其生物学特性进行研究。方法:运用正交法研究各因素对人羊膜上皮细胞分离、培养及冻存指标的影响,极差法分析数据得到优化的分离、培养与冻存工艺条件。对人羊膜上皮细胞进行原代与传代培养,通过镜下形态学观察,绘制细胞生长曲线,进行流式细胞术检测、免疫荧光染色及向肝样细胞分化等实验,观察人羊膜上皮细胞的生物学特性。结果与结论:(1)获得优化的人羊膜上皮细胞分离条件为:胰酶浓度0.25%,分4次消化,每次消化时间20 min;优化培养条件为:细胞接种浓度为4×108 L-1,表皮生长因子质量浓度为10μg/L,血清体积分数为5%;优化冻存条件为:细胞冻存浓度为1×1010 L-1,二甲基亚砜浓度为10%,血清体积分数为80%;(2)原代细胞在接种二三天内贴壁生长,贴壁后细胞呈不规则多角形,以铺路石样生长,传代后细胞贴壁与生长速度加快,冻存复苏的传代第2代细胞生长与扩增能力无明显下降;(3)免疫荧光染色显示,原代人羊膜上皮细胞强表达CK19、SSEA-4,不表达Vimentin、CD45和HLA-DR;原代与传代第4代免疫表型检测显示,人羊膜上皮细胞在培养传代过程中有上皮间充质转化现象发生;(4)向肝样细胞分化实验中,免疫荧光染色显示,诱导后的人羊膜上皮细胞肝细胞标志物白蛋白、CK18表达量明显上升,糖原染色显示诱导3周后的人羊膜上皮细胞有糖原合成;(5)结果表明,人羊膜上皮细胞易于获得且体外增殖能力强,表达胚胎干细胞保持未分化状态的表面标志物。
BACKGROUND:As current studies on isolation,culture and cryopreservation of human amniotic epithelial cells(h AECs) are relatively scattered,it is difficult to form a comprehensive and effective solution to meet the clinical needs of stem cells for transplantation in future.OBJECTIVE:To establish the technology of isolation,culture and cryopreservation of h AECs,and to study the biological characteristics of h AECs.METHODS:Orthogonal method was used to study the effects of different factors on the separation,culture and cryopreservation,and range method was adopted to analyze the data to optimize the separation,culture and cryopreservation.We performed cell primary and passage cultures,morphology observed by microscope,drawn cell growth curve and flow cytometry assay,immunofluorescence staining,hepatocyte like cell differentiation to study the biological characteristics of h AECs.RESULTS AND CONCLUSION:(1) The optimal h AECs separation conditions were as follows:trypsin digestions were conducted at a concentration of 0.25%,four times,once for 20 minutes digestion;optimal conditions of culture were 4×108/L cell seeding density,10 μg/L epidermal growth factor,5% serum;optimal conditions of cryopreservation were 1×1010/L cell cryopreservation density,10% dimethyl sulfoxide,80% serum.(2) The primary cells were adhered to the wall in 2-3 days,exhibiting irregular polygon,paving stone-like growth.Cell adherence and growth rate were accelerated after subculture,and the growth and proliferation ability of passage 2 cells were not significantly decreased after cryopreservation and resuscitation.(3) Immunofluorescence staining showed that the primary cells strongly expressed SSEA-4 and CK19,but did not express Vimentin,CD45 and HLA-DR.The immunophenotype statistics of the primary and passage 4 cells showed the epithelial mesenchymal transition of h AECs in culture process.(4) Immunofluorescence staining showed that the liver cell marker expression of ALB,CK18 was significantly increased after h AECs were induced to differentiate into hepatocyte-like cells.Glycogen staining revealed glycogen synthesis in h AECs after 3 weeks of induction.To conclude,h AECs are easy to obtain and have strong proliferation ability in vitro,and express surface markers for undifferentiated embryonic stem cells.
BACKGROUND:As current studies on isolation,culture and cryopreservation of human amniotic epithelial cells(h AECs) are relatively scattered,it is difficult to form a comprehensive and effective solution to meet the clinical needs of stem cells for transplantation in future.OBJECTIVE:To establish the technology of isolation,culture and cryopreservation of h AECs,and to study the biological characteristics of h AECs.METHODS:Orthogonal method was used to study the effects of different factors on the separation,culture and cryopreservation,and range method was adopted to analyze the data to optimize the separation,culture and cryopreservation.We performed cell primary and passage cultures,morphology observed by microscope,drawn cell growth curve and flow cytometry assay,immunofluorescence staining,hepatocyte like cell differentiation to study the biological characteristics of h AECs.RESULTS AND CONCLUSION:(1) The optimal h AECs separation conditions were as follows:trypsin digestions were conducted at a concentration of 0.25%,four times,once for 20 minutes digestion;optimal conditions of culture were 4×108/L cell seeding density,10 μg/L epidermal growth factor,5% serum;optimal conditions of cryopreservation were 1×1010/L cell cryopreservation density,10% dimethyl sulfoxide,80% serum.(2) The primary cells were adhered to the wall in 2-3 days,exhibiting irregular polygon,paving stone-like growth.Cell adherence and growth rate were accelerated after subculture,and the growth and proliferation ability of passage 2 cells were not significantly decreased after cryopreservation and resuscitation.(3) Immunofluorescence staining showed that the primary cells strongly expressed SSEA-4 and CK19,but did not express Vimentin,CD45 and HLA-DR.The immunophenotype statistics of the primary and passage 4 cells showed the epithelial mesenchymal transition of h AECs in culture process.(4) Immunofluorescence staining showed that the liver cell marker expression of ALB,CK18 was significantly increased after h AECs were induced to differentiate into hepatocyte-like cells.Glycogen staining revealed glycogen synthesis in h AECs after 3 weeks of induction.To conclude,h AECs are easy to obtain and have strong proliferation ability in vitro,and express surface markers for undifferentiated embryonic stem cells.
引文
[1]Marongiu F,Gramignoli R,Dorko K,et al.Hepatic Differentiation of Amniotic Epithelial Cells.Hepatology.2011;53(5):1719-1729.
[2]Niknejad H,Yazdanpanah G.Opposing effect of amniotic membrane on angiogenesis originating from amniotic epithelial cells.Ira J Med Hypotheses Ideas.2014;8(1):39-41.
[3]Toshio M,Fabio M,Ewa E,et al.Isolation of amniotic epithelial stem cells.Curr Protoc Stem Cell Biol.2010;Chapter 1:Unit 1E.3.
[4]Sean M,Sharina R,Rutu A,et al.Amnion Epithelial Cell Isolation and Characterization for Clinical Use.Curr Protoc Stem Cell Biol.2010;Chapter 1:1E.6.1-1E.6.25.
[5]Gita P,Vijesh V,Yang TJ,et al.Changes in culture expanded human amniotic epithelial cells:implications for potential therapeutic applications.Pl Os One.2011;6(11):e26136-e26136.
[6]Adinolfi M,Akle CA,Mccoll I,et al.Expression of HLA antigens,beta 2-microglobulin and enzymes by human amniotic epithelial cells.Nature.1982;295(5847):325-327.
[7]Satoko Y,Kunihiko T,Yasuharu U,et al.Differentiation of adult hepatic stem-like cells into pancreatic endocrine cells.Cell Transplant.2005;14(9):647-653.
[8]Tomoharu T,Isamu I,Shigeo S.Establishment and characterization of a pluripotent stem cell line derived from human amniotic membranes and initiation of germ layers in vitro.Human Cell.2004;17(3):125-130.
[9]程金华,朱化彬,孙秀柱,等.冷冻保护剂和胎牛血清对牛成纤维细胞冷冻效果的影响[J].中国畜牧杂志,2006,42(21):12-14.
[10]杨银芬,考桂兰,侯先志,等.不同细胞密度冻存对奶牛乳腺上皮细胞的影响[J].中国细胞生物学学报,2010,32(4):626-629.
[11]郭海平,郭冠萍,郝鹏.BHK21细胞冻存密度与复苏后活力的关系分析[J].畜牧与饲料科学,2010,31(3):6-7.
[12]陈露萍,王璞,汤海亮,等.不同冻存液对人诱导性多能干细胞冻存与复苏效果的影响[J].中国临床神经科学,2012,20(5):494-499.
[13]杨东斌,宋来君,杨波,等.不同人羊膜间充质干细胞冻存液冻存细胞效果比较[J].郑州大学学报:医学版,2009,44(3):568-572.
[14]金玲,陈剑,吴静,等.人羊膜上皮细胞的分离、纯化及其生物学特性研究[J].广东医学,2010,31(16):2055-2057.
[15]Paolo C,Chiara M,Pasquale V.Amniotic membrane transplantation in human immunodeficiency virus-positive children.Arch Ophthalmol.2008;126(6):866-867.
[16]方宁,宋秀军,张路,等.人羊膜上皮细胞的分离、培养及鉴定[J].遵义医学院学报,2009,32(2):121-124.
[17]Tabatabaei M,Mosaffa N,Nikoo S,et al.Isolation and partial characterization of human amniotic epithelial cells:the effect of trypsin.Avicenna J Med Biotechnol.2014;6(1):10-20.
[18]Liu X,Zhou Q,Zhang X,et al.In vitro culture and molecular characterization of human amniotic epithelial cells.Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi.2014;30(12):1318-1321.
[19]Yang TJ,Vijesh V,Han L Y,et al.Immunogenicity and immunomodulatory properties of hepatocyte-like cells derived from human amniotic epithelial cells.Curr Stem Cell Res Ther.2012;8(1):91-99.
[20]Miki T,Wong W,Zhou E,et al.Biological impact of xeno-free chemically defined cryopreservation medium on amniotic epithelial cells.Stem Cell Res Ther.2016;7(1):8.
[21]李云剑,林樾,燕辛,等.培养基血清浓度对人表皮干细胞增殖分化的影响[J].现代生物医学进展,2010,10(20):3831-3833.
[22]Koike C,Zhou K,Takeda Y,et al.Characterization of amniotic stem cells.Cell Reprogram.2014;16(4):298-305.
[23]Roy R,Kukucka M,Messroghli D,et al.Epithelial-tomesenchymal transition enhances the cardioprotective capacity of human amniotic epithelial cells.Cell Transplant.2013;24(6):985-1002.
[24]Elwan MA,Ishii T,Sakuragawa N.Characterization of dopamine D-2 receptor gene expression and binding sites in human placenta amniotic epithelial cells.Placenta.2003;24(6):658-663.
[25]Marcus AJ,Coyne TM,Black IB,et al.Fate of amnion-derived stem cells transplanted to the fetal rat brain:migration,survival and differentiation.J Cell Mol Med.2008;12(4):1256-1264.
[26]Wang JP.Phenotypic identification and differentiation potential analysis of two kinds of human amniotic cells.Zhongguo Shi Yan Xue Ye Xue Za Zhi.2012;20(1):146-53.
[27]Tan Jean L,Chan Siow T,Lo Camden Y,et al.Amnion cell-mediated immune modulation following bleomycin challenge:controlling the regulatory T cell response.Stem Cell Res Ther.2015;6:8.
[28]García-López G,García-Castro IL,Avila-González D,et al.Human Amniotic Epithelium(HAE)as a Possible Source of Stem Cells(SC).Gaceta Medica De Mexico.2015;151(1):66-74.
[29]Toshio M.Amnion-derived stem cells:In quest of clinical applications.Stem Cell Res Ther.2011;2(3):1-11.
[30]Drews K,Jozefczuk J,Prigione A,et al.Human induced pluripotent stem cells-from mechanisms to clinical applications.J Mol Med.2012;90(7):735-745.
[31]耿娟娟,崔勇,邱书奇.羊膜上皮细胞的体外培养及干细胞特性研究[J].实用医学杂志,2012,28(2):172-174.
[32]Okere B,Alviano F,Costa R,et al.In vitro differentiation of human amniotic epithelial cells into insulin-producing 3Dspheroids.Int J Immunopathol Pharmacol.2015;28(3):390-402.
[33]Kim S,Kim HS,Lee E,et al.In vivo hepatic differentiation potential of human cord blood-derived mesenchymal stem cells.Int J Mol Med.2011;27(5):701-706.
[34]Forte G,Minieri M,Cossa P,et al.Hepatocyte growth factor effects on mesenchymal stem cells:proliferation,migration,and differentiation.Stem Cells.2006;24(1):23-33.
[35]Kang NH,Hwang KA,Kim SU,et al.Potential antitumor therapeutic strategies of human amniotic membrane and amniotic fluid-derived stem cells.Cancer Gene Ther.2012;19(8):517-522.
[36]Lai D,Wang Y,Sun J,et al.Derivation and characterization of human embryonic stem cells on human amnion epithelial cells.Sci Rep.2015;5:10014.
[37]Tan JL,Chan ST,Wallace EM,et al.Human amnion epithelial cells mediate lung repair by directly modulating macrophage recruitment and polarization.Cell Transplant.2014;23(3):319-328.
[38]Carbone A,Paracchini V,Castellani S,et al.Human amnion-derived cells:prospects for the treatment of lung diseases.Curr Stem Cell Res Ther.2014;9(4):297-305.
[39]Hassan N,Ghasem Y.Anticancer effects of human amniotic membrane and its epithelial cells.Med Hypotheses.2014;82(4):488-489.
[40]Mc Donald CA,Payne NL,Sun G,et al.Immunosuppressive potential of human amnion epithelial cells in the treatment of experimental autoimmune encephalomyelitis.JNeuroinflammation.2015;12:112.
[41]Grzywocz Z,Pius-Sadowska E,Klos P,et al.Growth factors and their receptors derived from human amniotic cells in vitro.Folia Histochem Cytobiol.2014;52(3):163-170.
[2]Niknejad H,Yazdanpanah G.Opposing effect of amniotic membrane on angiogenesis originating from amniotic epithelial cells.Ira J Med Hypotheses Ideas.2014;8(1):39-41.
[3]Toshio M,Fabio M,Ewa E,et al.Isolation of amniotic epithelial stem cells.Curr Protoc Stem Cell Biol.2010;Chapter 1:Unit 1E.3.
[4]Sean M,Sharina R,Rutu A,et al.Amnion Epithelial Cell Isolation and Characterization for Clinical Use.Curr Protoc Stem Cell Biol.2010;Chapter 1:1E.6.1-1E.6.25.
[5]Gita P,Vijesh V,Yang TJ,et al.Changes in culture expanded human amniotic epithelial cells:implications for potential therapeutic applications.Pl Os One.2011;6(11):e26136-e26136.
[6]Adinolfi M,Akle CA,Mccoll I,et al.Expression of HLA antigens,beta 2-microglobulin and enzymes by human amniotic epithelial cells.Nature.1982;295(5847):325-327.
[7]Satoko Y,Kunihiko T,Yasuharu U,et al.Differentiation of adult hepatic stem-like cells into pancreatic endocrine cells.Cell Transplant.2005;14(9):647-653.
[8]Tomoharu T,Isamu I,Shigeo S.Establishment and characterization of a pluripotent stem cell line derived from human amniotic membranes and initiation of germ layers in vitro.Human Cell.2004;17(3):125-130.
[9]程金华,朱化彬,孙秀柱,等.冷冻保护剂和胎牛血清对牛成纤维细胞冷冻效果的影响[J].中国畜牧杂志,2006,42(21):12-14.
[10]杨银芬,考桂兰,侯先志,等.不同细胞密度冻存对奶牛乳腺上皮细胞的影响[J].中国细胞生物学学报,2010,32(4):626-629.
[11]郭海平,郭冠萍,郝鹏.BHK21细胞冻存密度与复苏后活力的关系分析[J].畜牧与饲料科学,2010,31(3):6-7.
[12]陈露萍,王璞,汤海亮,等.不同冻存液对人诱导性多能干细胞冻存与复苏效果的影响[J].中国临床神经科学,2012,20(5):494-499.
[13]杨东斌,宋来君,杨波,等.不同人羊膜间充质干细胞冻存液冻存细胞效果比较[J].郑州大学学报:医学版,2009,44(3):568-572.
[14]金玲,陈剑,吴静,等.人羊膜上皮细胞的分离、纯化及其生物学特性研究[J].广东医学,2010,31(16):2055-2057.
[15]Paolo C,Chiara M,Pasquale V.Amniotic membrane transplantation in human immunodeficiency virus-positive children.Arch Ophthalmol.2008;126(6):866-867.
[16]方宁,宋秀军,张路,等.人羊膜上皮细胞的分离、培养及鉴定[J].遵义医学院学报,2009,32(2):121-124.
[17]Tabatabaei M,Mosaffa N,Nikoo S,et al.Isolation and partial characterization of human amniotic epithelial cells:the effect of trypsin.Avicenna J Med Biotechnol.2014;6(1):10-20.
[18]Liu X,Zhou Q,Zhang X,et al.In vitro culture and molecular characterization of human amniotic epithelial cells.Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi.2014;30(12):1318-1321.
[19]Yang TJ,Vijesh V,Han L Y,et al.Immunogenicity and immunomodulatory properties of hepatocyte-like cells derived from human amniotic epithelial cells.Curr Stem Cell Res Ther.2012;8(1):91-99.
[20]Miki T,Wong W,Zhou E,et al.Biological impact of xeno-free chemically defined cryopreservation medium on amniotic epithelial cells.Stem Cell Res Ther.2016;7(1):8.
[21]李云剑,林樾,燕辛,等.培养基血清浓度对人表皮干细胞增殖分化的影响[J].现代生物医学进展,2010,10(20):3831-3833.
[22]Koike C,Zhou K,Takeda Y,et al.Characterization of amniotic stem cells.Cell Reprogram.2014;16(4):298-305.
[23]Roy R,Kukucka M,Messroghli D,et al.Epithelial-tomesenchymal transition enhances the cardioprotective capacity of human amniotic epithelial cells.Cell Transplant.2013;24(6):985-1002.
[24]Elwan MA,Ishii T,Sakuragawa N.Characterization of dopamine D-2 receptor gene expression and binding sites in human placenta amniotic epithelial cells.Placenta.2003;24(6):658-663.
[25]Marcus AJ,Coyne TM,Black IB,et al.Fate of amnion-derived stem cells transplanted to the fetal rat brain:migration,survival and differentiation.J Cell Mol Med.2008;12(4):1256-1264.
[26]Wang JP.Phenotypic identification and differentiation potential analysis of two kinds of human amniotic cells.Zhongguo Shi Yan Xue Ye Xue Za Zhi.2012;20(1):146-53.
[27]Tan Jean L,Chan Siow T,Lo Camden Y,et al.Amnion cell-mediated immune modulation following bleomycin challenge:controlling the regulatory T cell response.Stem Cell Res Ther.2015;6:8.
[28]García-López G,García-Castro IL,Avila-González D,et al.Human Amniotic Epithelium(HAE)as a Possible Source of Stem Cells(SC).Gaceta Medica De Mexico.2015;151(1):66-74.
[29]Toshio M.Amnion-derived stem cells:In quest of clinical applications.Stem Cell Res Ther.2011;2(3):1-11.
[30]Drews K,Jozefczuk J,Prigione A,et al.Human induced pluripotent stem cells-from mechanisms to clinical applications.J Mol Med.2012;90(7):735-745.
[31]耿娟娟,崔勇,邱书奇.羊膜上皮细胞的体外培养及干细胞特性研究[J].实用医学杂志,2012,28(2):172-174.
[32]Okere B,Alviano F,Costa R,et al.In vitro differentiation of human amniotic epithelial cells into insulin-producing 3Dspheroids.Int J Immunopathol Pharmacol.2015;28(3):390-402.
[33]Kim S,Kim HS,Lee E,et al.In vivo hepatic differentiation potential of human cord blood-derived mesenchymal stem cells.Int J Mol Med.2011;27(5):701-706.
[34]Forte G,Minieri M,Cossa P,et al.Hepatocyte growth factor effects on mesenchymal stem cells:proliferation,migration,and differentiation.Stem Cells.2006;24(1):23-33.
[35]Kang NH,Hwang KA,Kim SU,et al.Potential antitumor therapeutic strategies of human amniotic membrane and amniotic fluid-derived stem cells.Cancer Gene Ther.2012;19(8):517-522.
[36]Lai D,Wang Y,Sun J,et al.Derivation and characterization of human embryonic stem cells on human amnion epithelial cells.Sci Rep.2015;5:10014.
[37]Tan JL,Chan ST,Wallace EM,et al.Human amnion epithelial cells mediate lung repair by directly modulating macrophage recruitment and polarization.Cell Transplant.2014;23(3):319-328.
[38]Carbone A,Paracchini V,Castellani S,et al.Human amnion-derived cells:prospects for the treatment of lung diseases.Curr Stem Cell Res Ther.2014;9(4):297-305.
[39]Hassan N,Ghasem Y.Anticancer effects of human amniotic membrane and its epithelial cells.Med Hypotheses.2014;82(4):488-489.
[40]Mc Donald CA,Payne NL,Sun G,et al.Immunosuppressive potential of human amnion epithelial cells in the treatment of experimental autoimmune encephalomyelitis.JNeuroinflammation.2015;12:112.
[41]Grzywocz Z,Pius-Sadowska E,Klos P,et al.Growth factors and their receptors derived from human amniotic cells in vitro.Folia Histochem Cytobiol.2014;52(3):163-170.