DNA损伤在去分化源性表皮干细胞安全性评价中的作用及相关机制的研究  

Safety evaluation of dedifferentiation-derived epidermal stem cells after damage to DNA and investigation of related molecular mechanisms

作　　者：潘宇[1] 余细勇[1] 吴漫茵 付小兵[3] 蔡飒[2] 

机构地区：[1]广东省人民医院广东省医学科学院医学研究中心,广东广州510080 [2]深圳大学医学部,广东深圳518060 [3]解放军总医院第一附属医院全军创伤修复与组织再生重点实验室暨皮肤损伤修复与组织再生北京市重点实验室,北京100048

出　　处：《感染．炎症．修复》2015年第1期17-22,共6页Infection Inflammation Repair

基　　金：国家自然科学基金资助项目(81000011;81272080;81000835);深圳市科技研发资金基础研究计划(JC201005280429A;JCYJ20120613101917373)

摘　　要：目的:从分子、细胞及在体水平评估DNA损伤和非DNA损伤因素诱导所得去分化源性表皮干细胞的安全性,探讨DNA损伤及其应答分子对其安全性评估的意义与机制。方法:分别采用紫外线(UV)照射和bFGF处理诱导方案诱导表皮细胞去分化。采用MTT法比较正常培养和血清培养条件下的两组去分化源性表皮干细胞的增殖能力;流式细胞术检测凋亡情况;Western blotting检测各组去分化过程中DNA损伤应答分子γ-H2AX、激活型caspase、p53、p21表达的变化;在雌性BALB/c裸小鼠右侧腋窝皮下注射人黑色素瘤细胞及两种去分化源性表皮干细胞,同时设生理盐水注射对照,30d后取瘤块称重,评估两种去分化源性干细胞的成瘤性。结果:1两种去分化源性表皮干细胞均表现出较强的增殖能力,UV照射所得干细胞具有较强的血清耐受性;2UV照射可导致表皮细胞DNA损伤和细胞凋亡,影响表皮细胞中p53和p21水平,而bFGF处理组无此作用;3两种去分化源性表皮干细胞均无体内成瘤性。结论:去分化过程中细胞DNA损伤与否是评价去分化源性干细胞安全性的较敏感的关键指标,因此bFGF处理诱导与UV照射所得的表皮干细胞相比更为安全可靠。Objective：To evaluate the safety of the dedifferentiation-derived epidermal stem cells generated either by damage to DNA or non-DNA-damage with protocols of molecular,cellular and in vivo study,and to investigate the mechanisms of DNAdamage responsers in the process of epidermal cells dedifferentiation.Methods：Two types of dedifferentiation-derived epidermal stem cell were generated individually using the ultraviolet（UV）radiation or basic fibroblast growth factor（bFGF）treatment protocol.The proliferation ability and serum tolerance of different dedifferentiation-derived epidermal stem cells were compared by MTT assay.Apoptosis of dedifferentiated cells was detected by flow cytometry.The changes in occurrence of DNA damage and related molecular elements（phospho-histoneγ-H2 AX,cleaved caspase,p53,p21）were detected by Western blotting.Different epidermal stem cells,human melanoma cells,or normal saline were injected subcutaneously into right armpits of female nude mice,and their tumorigenic potential was assessed.Results：1The proliferation of dedifferentiation-derived epidermal stem cells were increased in either UV radiation or bFGF-treated group,while cells from UV radiation group appeared to be more tolerant to serum than those from bFGF-treated group.2 UV radiation,but not bFGF treatment,caused DNA damage and apoptosis in epidermal cells and the levels of p53 and p21were increased.3 No tumor formation was found in dedifferentiation-derived epidermal stem cells of UV radiation nor bFGF-treated group.Conclusions：The occurrence of DNA damage in the process of dedifferentiation is sensitive and crucial in evaluating the safety of dedifferentiation-derived epidermal stem cells.The induced epidermal stem cells from bFGF-treated group are more reliable than those from UV radiation group.

关 键 词：表皮干细胞 去分化 DNA 损伤 安全性评价 

分 类 号：R361.3[医药卫生—病理学]