【摘要】：背景:目前人羊膜上皮細胞分離、培養(yǎng)及凍存的研究較為分散,難以形成全面有效的方案以滿足未來對移植用干細胞的臨床需求。目的:建立人羊膜上皮細胞優(yōu)化的分離、培養(yǎng)與凍存工藝,并對其生物學(xué)特性進行研究。方法:運用正交法研究各因素對人羊膜上皮細胞分離、培養(yǎng)及凍存指標的影響,極差法分析數(shù)據(jù)得到優(yōu)化的分離、培養(yǎng)與凍存工藝條件。對人羊膜上皮細胞進行原代與傳代培養(yǎng),通過鏡下形態(tài)學(xué)觀察,繪制細胞生長曲線,進行流式細胞術(shù)檢測、免疫熒光染色及向肝樣細胞分化等實驗,觀察人羊膜上皮細胞的生物學(xué)特性。結(jié)果與結(jié)論:(1)獲得優(yōu)化的人羊膜上皮細胞分離條件為:胰酶濃度0.25%,分4次消化,每次消化時間20 min;優(yōu)化培養(yǎng)條件為:細胞接種濃度為4×108 L-1,表皮生長因子質(zhì)量濃度為10μg/L,血清體積分數(shù)為5%;優(yōu)化凍存條件為:細胞凍存濃度為1×1010 L-1,二甲基亞砜濃度為10%,血清體積分數(shù)為80%;(2)原代細胞在接種二三天內(nèi)貼壁生長,貼壁后細胞呈不規(guī)則多角形,以鋪路石樣生長,傳代后細胞貼壁與生長速度加快,凍存復(fù)蘇的傳代第2代細胞生長與擴增能力無明顯下降;(3)免疫熒光染色顯示,原代人羊膜上皮細胞強表達CK19、SSEA-4,不表達Vimentin、CD45和HLA-DR;原代與傳代第4代免疫表型檢測顯示,人羊膜上皮細胞在培養(yǎng)傳代過程中有上皮間充質(zhì)轉(zhuǎn)化現(xiàn)象發(fā)生;(4)向肝樣細胞分化實驗中,免疫熒光染色顯示,誘導(dǎo)后的人羊膜上皮細胞肝細胞標志物白蛋白、CK18表達量明顯上升,糖原染色顯示誘導(dǎo)3周后的人羊膜上皮細胞有糖原合成;(5)結(jié)果表明,人羊膜上皮細胞易于獲得且體外增殖能力強,表達胚胎干細胞保持未分化狀態(tài)的表面標志物。
[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.
【作者單位】： 浙江省臍帶血造血干細胞庫協(xié)和華東干細胞基因工程有限公司;
【基金】：浙江省網(wǎng)上技術(shù)市場交易產(chǎn)業(yè)化項目(2012jssc02)~~
【分類號】：R329.2

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