低温冻存对人脂肪来源干细胞成骨能力影响的实验研究  

EFFECT OF CRYOPRESERVATION ON GROWTH AND OSTEOGENESIS OF HUMAN ADIPOSE-DERIVED STEM CELLS

作　　者：刘广鹏[1] 李宇琳[1] 孙剑[1] 周恒[1] 崔磊[1] 

机构地区：[1]上海交通大学医学院附属第九人民医院整复外科组织工程国家工程研究中心,上海200241

出　　处：《中国修复重建外科杂志》2010年第10期1224-1227,共4页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：国家高技术研究发展计划(863)资助项目(2006AA02A123);国家自然科学基金资助项目(30800282);上海市科技启明星跟踪计划资助项目(10QH1402400)~~

摘　　要：目的脂肪来源干细胞(adipose-derived stem cells,ADSCs)是组织工程种子细胞的重要来源之一。探讨低温保存对hADSCs体外生长特性及成骨分化潜能的影响,验证冻存后的ADSCs作为组织工程骨种子细胞的可行性。方法取自愿捐献的经脂肪抽吸术获取人脂肪组织,采用胶原酶消化法分离hADSCs。取第2代hADSCs置于-196℃液氮保存4周后,37℃复苏并测定细胞存活率。实验分为两组,实验组为低温保存的hADSCs,对照组为未经低温保存的hADSCs;均用成骨诱导液诱导培养。采用DNA定量(Hoechst33258荧光比色法)测定细胞体外增殖活性,ALP染色及茜素红染色法测定ALP活性和Ca2+含量,观察低温冻存对细胞成骨能力的影响。结果低温冻存复苏后的hADSCs存活率为90.44%±2.62%。两组细胞数量随成骨诱导时间延长不断增加,7d达平台期;ALP表达活性也不断增加,于成骨诱导11d达平台期,且2周时ALP染色均呈阳性;成骨诱导3周时两组茜素红染色均呈强阳性反应。两组各时间点细胞数量、ALP活性和Ca2+含量比较差异均无统计学意义(P>0.05)。结论低温保存的hADSCs体外生长及成骨能力未发生显著变化,可以作为组织工程骨的种子细胞。Objective As one of the adult stem cells, adipose-derived stem cells （ADSCs） have become an important seed cell source for tissue engineering recently. But whether the thawed cryopreserved ADSCs could be used to tissue engineered bone remains unknown. To investigate the effect of cryopreservation on the growth and osteogenesis of ADSCs in vitro. Methods The ADSCs were isolated from the adipose aspirates by collagenase digestion method. For the experimental group, the 2nd generation cells were stored with a simple method of cryopreservation by slow cooling with dimethyl sulphoxide as a cryoprotectant and rapid thawing. After cryopreserved in liquid nitrogen for 4 weeks, ADSCs were recovered and cultured in osteogenic media, with non-cryopreserved ADSCs as the control group. The osteogenic differentiation was evaluated by alkaline phosphatase （ALP） staining and Alizarin red O staining at 2 and 3 weeks respectively. The cell growth and osteogenesis of ADSCs were further determined using DNA assay and the ALP activity and calcium content were measured. Results The survival percentage of the cryopreserved cells was 90.44% _＋ 2.62%. The cell numbers and ALP activity increased with osteogenic induction time, and reach plateaus at 7 days and 11 days, respectively. The ALP staining and Alizarin red O staining results were both positive at 2 weeks and 3 weeks after osteogenic induction, respectively. And no significant difference in the cells number, ALP activity, and calcium content were found between experimental group and control group （P 〉 0.05）. Conclusion Cryopreservation does not affect the growth and osteogenesis of ADSCs, and the cryopreserved ADSCs can be used as cell source for tissue engineered bone.

关 键 词：组织工程 低温冻存 脂肪来源干细胞 成骨分化 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]