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血小板第4因子对急性放射损伤小鼠骨髓基质细胞

2014-09-26 01:00 作者：高瑛，杨岚，田琼，方恒虎，聂蕾，刘水冰，卫张蕊
【关键词】 骨髓基质细胞
Protective effects of platelet factor 4 on bone marrow stromal cells of acute radiation injury in mice
　　【Abstract】 AIM: To investigate the protective effects of platelet factor 4 (PF4) on bone marrow stromal cells (BMSCs) of radiated mice and to study the protective mechanism of hematopoietic radiation injury in mice. METHODS: Forty male mice were randomized to four groups (R, R P, N P and N) and two of groups (R, R P) were exposed wholebodily to 5.0 Gy 60Co γrays. The mice were treated with injections of PF4 (50μg/ kg), ip twice at an interval of 6 h and 20 h. After the second injection, they were irradiated. The cell cycle and apoptosis of bone marrow stromal cells (BMSCs) cultured in vitro after radiation were analyzed by FACS after ten days culture. The expression of p27kip1 in the cells was detected using immunohistochemistry. RESULTS: The 24th hour adhesion rate of BMSC in radiated mice respectively reached 37%, 58%, 74% and 79.3%. The cell percentage of G0 G1 phase increased and those of G2 M decreased in R group, compared with those in R P, N P and N groups. But the DNA content of the radiated group of BMSC decreased remarkably compared with those in the other three groups. The expression of p27kip1 was downregulated compared with that of BMSC without PF4 protection. CONCLUSION: The protective effects of PF4 on BMSCs with radiated injury probably result from the inhibition of the expression of p27kip1.
　　【Keywords】 marrow stromal cells; platelet factor 4; radiation injuries; radiatior protection; p27
　　【摘要】 目的： 探讨血小板第4因子(platelet factor 4, PF4)对小鼠5.0 Gy γ射线全身照射后骨髓基质细胞（bone marrow stromal cell, BMSC）的保护作用，进一步探讨PF4对造血的辐射防护机制. 方法： 40只雄性小鼠随机分为4组： ① 单纯放射组（R），② 放射＋PF4组（R P），③ 正常＋PF4组（N P），④ 正常对照组（N）. 小鼠照射前分别于26和20 h ip PF4，每次剂量50 μg/ kg. 于照射后3 d取BMSC体外培养，倒置显微镜下观察贴壁细胞生长状况，并于培养10 d后用流式细胞仪测定贴壁细胞的细胞周期和DNA含量，免疫组化（SP法）检测细胞p27kip1表达. 结果： 放射损伤小鼠BMSC 24 h贴壁率4组依次为37％, 58%, 74%, 79.3%；流式细胞仪检测结果表明R组G0 G1期细胞显著高于其余3组，而G2 M期细胞显著低于其余三组，DNA含量显著低于其余3组；R组p27kip1表达明显强于其余3组. 结论： PF4对放射损伤的BMSC有保护作用，可能与抑制p27kip1表达有关.
　　【关键词】 骨髓基质细胞；血小板因子4；辐射损伤；辐射防护；p27
　
　　0引言

　　造血功能障碍是放射损伤时最基本的病理变化. 此时的造血功能障碍涉及造血干/祖细胞和造血基质细胞损伤. 骨髓基质细胞(bone marrow stromal cell, BMSC)是造血诱导微环境(hematopoietic inductive microenvironment, HIM)的重要组成成分. 血小板第4因子(PF4)能可逆地抑制造血干细胞、祖细胞的生长，又是骨髓细胞调节的负调节因子,并因此而保护骨髓造血干祖细胞免受辐射和细胞毒剂的损伤［1，2］. 在本实验中我们首次以体外培养放射损伤小鼠的BMSC为基础，观察PF4对其的保护作用，进一步探讨PF4对放射损伤的保护机制.
　　1材料和方法
　　1.1材料
　　BABL/c健康雄性小鼠40只，体质量22~24 g，第四军医大学实验动物中心提供；RPMI1640培养基（Gibco）；PF4（Sigma）：用双蒸水稀释成工作液,置4℃冰箱保存备用,稀释浓度为100 mg/L；特级小牛血清、马血清（杭州，四季青）；胰酶（Sigma）；P27kip1, SP试剂盒及DAB显色试剂盒（武汉博士德）.