人羊膜上皮细胞培养工艺的优化及生物学特性

发布时间：2018-07-21 19:19

【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。
[Abstract]:Background: at present, the study of human amniotic epithelial cells isolation, culture and cryopreservation is scattered, it is difficult to form a comprehensive and effective program to meet the future clinical needs of transplantation stem cells. Aim: to establish the optimized separation, culture and cryopreservation of human amniotic epithelial cells and to study their biological characteristics. Methods: orthogonal method was used to study the effects of various factors on the separation, culture and freezing of human amniotic epithelial cells. The optimum separation, culture and freezing conditions were obtained by using the range method. The primary and passage culture of human amniotic epithelial cells was carried out. The growth curve of human amniotic epithelial cells was measured by flow cytometry, immunofluorescence staining and hepatoid cell differentiation. To observe the biological characteristics of human amniotic epithelial cells. Results and conclusion: (1) the optimized conditions for the isolation of human amniotic epithelial cells were as follows: the concentration of trypsin was 0.25 and the cells were digested four times. The optimum conditions were as follows: cell inoculation concentration was 4 脳 108L ~ (-1), epidermal growth factor concentration was 10 渭 g / L, serum volume fraction was 5 ~ (th). The optimal conditions were as follows: cell freezing concentration was 1 脳 1010 L ~ (-1), dimethyl sulfoxide concentration was 1 脳 10 ~ (-1), dimethyl sulfoxide (DMSO) concentration was 1 脳 1010 L ~ (-1). The volume fraction of serum was 80. (2) the primary cells adhered to the wall within two or three days of inoculation. The adherent cells showed irregular polygonal shape, which grew like paving stone. After passage, the adhesion and growth rate of the cells were accelerated, and the ability of growth and expansion of the second passage of cryopreservation and resuscitation was not significantly decreased. (3) Immunofluorescence staining showed that the growth and expansion of the cells in the second passage were not significantly decreased. The primary human amniotic epithelial cells strongly expressed CK19mSSEA-4, but not VimentinCCD45 and HLA-DR.The primary and passage 4 immunophenotypic tests showed that the epithelial mesenchymal transformation occurred during the passage of human amniotic epithelial cells. (4) in the differentiation experiment of hepatoid cells, human amniotic epithelial cells were transformed into hepatocytes. Immunofluorescence staining showed that the expression of albumin CK18 in human amniotic epithelial cells increased significantly, and glycogen staining showed glycogen synthesis in human amniotic epithelial cells 3 weeks after induction. (5) the results showed that Human amniotic epithelial cells are easy to obtain and have strong proliferative ability in vitro, and express surface markers of embryonic stem cells maintaining undifferentiated state.
【作者单位】：浙江省脐带血造血干细胞库协和华东干细胞基因工程有限公司;
【基金】：浙江省网上技术市场交易产业化项目(2012jssc02)~~
【分类号】：R329.2

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