低温保存的GFP标记脂肪源性干细胞体外构建组织工程骨的研究  

The effect of cryopreservation on the growth and osteogenesis capability of GFP-labeled canine adipose-derived stem cells on demineralized bone matrix

作　　者：刘广鹏[1,2] 孙剑[1,2] 李宇琳[1,2] 周恒[1,2] 崔磊[1,2] 

机构地区：[1]上海市第九人民医院整形外科组织工程重点实验室 [2]组织工程(上海)国家工程研究中心

出　　处：《中国医药生物技术》2011年第1期7-11,共5页Chinese Medicinal Biotechnology

基　　金：国家自然科学基金(30800282);上海市青年科技启明星跟踪计划(10QH1402400)

摘　　要：目的研究经低温保存的GFP基因转染的犬脂肪源性干细胞(ASCs)在脱钙骨(DBM)支架材料上的生长特性及成骨分化潜能。方法胶原酶消化法分离获取并常规培养犬ASCs,用绿色荧光蛋白(GFP)重组逆转录病毒载体转染第2代ASCs,然后进行低温保存。-196℃液氮保存4周后,37℃复苏,测定细胞存活率。低温保存前后的ASCs接种DBM,用成骨诱导液诱导培养2周。激光共聚焦显微镜观察细胞在DBM上的生长情况,DNA定量(Hoechst33258法)测定细胞的体外增殖活性。通过测定碱性磷酸酶(ALP)活性和骨钙蛋白(OCN)含量,观察低温冻存对细胞在支架材料上成骨能力的影响。结果低温冻存复苏后细胞的存活率>90%,激光共聚焦显微镜显示细胞在材料上黏附生长良好。体外培养12d时细胞增殖达到平台期,ALP活性与OCN含量则随培养时间延长而不断上升,低温保存前后细胞的检测结果无显著差异(P>0.05)。结论低温保存对GFP标记的犬ASCs在脱钙骨上的体外生长及成骨能力无显著影响,可以作为组织工程骨的种子细胞。Objective To investigate the effect of cryopreservation on the growth and osteogenesis of green fluorescent protein gene（GFP） labeled canine adipose-derived stem cells（ASCs） on demineralized bone matrix（DBM）.Methods Canine ASCs were isolated from the adipose tissue by collagenase digestion.Cells of passage 2 were infected with recombinant RV-GFP expression vector directly,and then cryopreserved in liquid nitrogen for 4 weeks.Cell recovery was measured after thawing.The cryopreserved and non-cryopreserved（control） ASCs were seeded onto DBM and cultured in osteogenic media for 2 weeks.Cell proliferation on the scaffolds was observed by laser confocal microscope and measured by DNA assay.The osteogenic differentiation was evaluated by alkaline phosphatase（ALP） activity and osteocalcin（OCN） content.Results The percentage of cell survival after thawing was above 90%.Cryopreserved cells adhered well to the DBM and grew rapidly.There were no difference in the values of DNA,ALP and OCN between the cryopreserved and control ASCs.Conclusion Cryopreservation does not affect the GFP expression,as well as the growth and osteogenesis of canine ASCs onto the DBM scaffolds,and the cryopreserved ASCs could be used as cell source for tissue engineered bone.

关 键 词：低温保存 绿色荧光蛋白质类 组织工程 成体干细胞 动物实验 

分 类 号：R318[医药卫生—生物医学工程]