解剖学报

解剖学报 ›› 2013, Vol. 44 ›› Issue (2 ): 189-193.doi: 10.3969/j.issn.0529-1356.2013.02.008

• 细胞和分子生物学 • 上一篇    下一篇

氧化低密度脂蛋白可促进小鼠造血干细胞衰老

张先平1 张贵海2  文坤明3  刘俊1  徐春燕1 王璐1  姜蓉1  王亚平1*    

  1. 1. 重庆医科大学干细胞与组织工程学研究室，组织学与胚胎学教研室，重庆400016; 2. 遵义医学院附属医院肿瘤科，贵州 遵义 563003; 3. 遵义医学院附属医院胃肠外科，贵州 遵义 563003
  • 收稿日期:2012-05-04 修回日期:2012-06-12 出版日期:2013-04-06 发布日期:2013-04-06
  • 基金资助:

    国家自然科学基金资助项目;自然科学基金资助项目

Oxidation low density lipoprotein promotes aging of mouse hematopoietic stem cells through regulating cell cycles

ZHANG Xian-ping1 ZHANG Gui-hai2 WEN Kun-ming3 LIU Jun1 XU Chu-yan1 WANG Lu1 JIANG Rong1 WANG Ya-ping 1*   

  1. 1.   Laboratory of Stem Cell and Tissue Engineering，Department of Histology and Embryology, Chongqing Medical University，Chongqing 400016，China; 2.Department of Oncology of Zunyi Medical Collage Affiliated Hospital, Guizhou Zunyi 563003, China; 3. Department of Gastroenterological Surgery, Zunyi Medical Collage Affiliated Hospital, Guizhou Zuny 563003, China
  • Received:2012-05-04 Revised:2012-06-12 Online:2013-04-06 Published:2013-04-06

摘要：

目的 观察氧化低密度脂蛋白（ox-LDL）对造血干细胞（HSCs）细胞周期调控基因p16、p21、CDK2、CDK4及细胞周期蛋白（cyclinD）表达的影响，探讨ox-LDL诱导HSCs衰老的可能分子机制。 方法 采用免疫磁性分选法分离纯化小鼠Sca-1+ HSCs，并与ox-LDL共培养，通过衰老相关β-半乳糖苷酶（SA-β-Gal）染色检测衰老HSCs，四唑氮盐（MTS）比色法与多向造血祖细胞（CFU-Mix）混合集落培养检测HSCs自我更新能力和多向分化潜能，流式细胞术分析细胞周期分布，荧光定量PCR及Western blotting检测HSCs p16、p21、CDK2、CDK4及cyclinD表达。 结果 与对照组比较，ox-LDL能增加 HSCs SA-β-Gal染色阳性细胞率；促进HSCs停滞于G1 期，G0/G1 期细胞增多、S 期细胞减少，减弱HSCs自我更新与多向分化能力； ox-LDL能上调HSCs p16和p21 mRNA及蛋白表达水平，下调CDK4及cyclinD蛋白表达，而对CDK2蛋白表达无影响。结论 ox-LDL能通过上调p16、p21表达及下调CDK4、cyclinD表达，在体外促进HSCs衰老。

关键词: 衰老 , 造血干细胞 , 氧化低密度脂蛋白 , 细胞周期 , 流式细胞术 , 小鼠

Abstract:

Objective To observe the effect of oxidation low density lipoprotein on aging of hematopoietic stem cells (HSCs) and to reveal the underlying mechanisms that ox-LDL induce the aging of HSCs. Method Mouse HSCs were isolated by magnetic cell sorting with Sca-1 staining. Sca-1+ HSCs were cultured with ox-LDL. Senescence-associated β-galactos idase (Saβ-Gal) staining was used to identify aging HSCs. The capacity of self-renewal of HSCs was evaluated by MTS assay. CFU-Mix cultivation was used to evaluate the potency of differentiation in HSCs. Cell cycles were detected by flow cytometry. The expressions of p16, p21, CDK4 and cyclinD were determined by realtime quantitative PCR (qRT-PCR) and Western blotting analysis. Results Exogenous ox-LDL significantly increased the number of Saβ-Gal staining postive in HSCs, promoted HSCs to arrest at S stage, elevated the ratio of G0/G1 stage , decreased the ratio of S stage and the number of CFU-Mix, and inhibited the proliferation of HSCs compared to HSCs without ox-LDL treatment group. Ox-LDLremarkedly upregulated the expression of p16 and p21 in mRNA and protein levels, decreased the protein expression of CDK4 and cyclinD. There was no significant difference in the protein expression of CDK2 in HSCs. Conclusion ox-LDL may induce mice HSCs aging through upregulating the expressions of p16 and p21, and downregulating CDK4 and cyclinD expressions.

Key words: Aging , Hematopoietic stem cell , Oxidation low density lipoprotein , Cell cycle , Flow cytomety , Mouse