人羊膜间充质干细胞无血清培养方法的建立  

Establishment of a serum-free culture method for human amnion mesenchymal stem cells

作　　者：刘晓玉 施萍 潘伯臣[1] 庞希宁[2,3] LIU Xiao-yu;SHI Ping;PAN Bo-chen;PANG Xi-ning(Center of Reproductive Medicine,Shengjing Hospital of China Medical University,Shenyang 110004;Department of Stem Cells and Regenerative Medicine,China Medical University,Shenyang 110122;Shenyang Amnion Bioengineering and Technology Research and Development Center Co.Ltd,Shenyang 110015,China)

机构地区：[1]中国医科大学附属盛京医院生殖医学中心,辽宁沈阳110004 [2]中国医科大学干细胞与再生医学研究室,辽宁沈阳110122 [3]沈阳艾米奥生物工程技术研发中心有限公司,辽宁沈阳110015

出　　处：《基础医学与临床》2021年第8期1181-1185,共5页Basic and Clinical Medicine

基　　金：沈阳市干细胞与再生医学重点实验室项目(20-204-4-31)。

摘　　要：目的建立一种人羊膜间充质干细胞(hAMSCs)的无血清培养方法。方法用胰蛋白酶-胶原蛋白酶两步消化法分离人羊膜间充质干细胞,用含1×ITS、0.5%人血清白蛋白、10 ng/mL bFGF、100μg/mL L-ascorbic acid、100 U/mL青链霉素及2.5μg/mL两性霉素B的DMEM/F12无血清培养基进行培养,倒置相差显微镜下观察细胞形态及增殖状态,免疫荧光技术检测细胞表型,WST法测定细胞增殖曲线,细胞划痕伤口闭合试验检测无血清培养的细胞迁移能力,实时定量PCR检测生长因子VEGF、KGF、PDGF和IGF-1的表达。结果无血清培养并纯化扩增的人羊膜间充质干细胞呈梭形,表达细胞表面标志物CD73和CD90,不表达CD45和CD34。细胞增殖曲线为S形,对数增殖期的平均倍增时间为72 h,与胎牛血清培养的人羊膜间充质干细胞相比,迁移能力增强,生长因子VEGF、KGF、PDGF和IGF-1表达上调(P<0.05)。结论建立了一种人羊膜间充质干细胞的无血清培养方法,为临床应用提供实验依据。Objective To establish a method of serum free culture for human amnion mesenthymal stem cells.Methods The hAMSCs were isolated by trypsin-collagenase two step digestion method,then cultured in serum free medium.Then immunofluorescence and WST assay were used to detect cell phenotype and cells growth.Cell migration of hAMSCs cultured in serum free medium was examined by scratch wound closure assay.The expression of growth factor was detected by real time PCR assay.Results The hAMSCs cultured in serum free medium exhibited spindle-shape characteristics,were positive for CD73,CD90,but negative for CD45 and CD34.The cell proliferation showed a U-shaped curve and the average doubling time of was 72 hours.The scratch wound closure assay demonstrated a statistically significant increase of migration as compared with those cultured in FBS.The expression of VEGF,KGF,PDGF,IGF-1 was up-regulated compared with those cultured in FBS.ConclusionsA serum free culture method of hAMSCs is established,which provides an experimental evidence supportingpotential application.

关 键 词：人羊膜间充质干细胞(hAMSCs) 无血清培养 增殖 迁移 

分 类 号：Q331[生物学—遗传学]