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血小板第4因子對(duì)急性放射損傷小鼠骨髓基質(zhì)細(xì)胞的保護(hù)作用
作者：高瑛，楊嵐，田瓊，方恒虎，聶蕾，劉水冰，衛(wèi)張蕊【關(guān)鍵詞】 骨髓基質(zhì)細(xì)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： 探討血小板第4因子(platelet factor 4, PF4)對(duì)小鼠5.0 Gy γ射線全身照射后骨髓基質(zhì)細(xì)胞（bone marrow stromal cell, BMSC）的保護(hù)作用，進(jìn)一步探討PF4對(duì)造血的輻射防護(hù)機(jī)制. 方法： 40只雄性小鼠隨機(jī)分為4組： ① 單純放射組（R），② 放射＋PF4組（R P），③ 正常＋PF4組（N P），④ 正常對(duì)照組（N）. 小鼠照射前分別于26和20 h ip PF4，每次劑量50 μg/ kg. 于照射后3 d取BMSC體外培養(yǎng)，倒置顯微鏡下觀察貼壁細(xì)胞生長狀況，并于培養(yǎng)10 d后用流式細(xì)胞儀測(cè)定貼壁細(xì)胞的細(xì)胞周期和DNA含量，免疫組化（SP法）檢測(cè)細(xì)胞p27kip1表達(dá). 結(jié)果： 放射損傷小鼠BMSC 24 h貼壁率4組依次為37％, 58%, 74%, 79.3%；流式細(xì)胞儀檢測(cè)結(jié)果表明R組G0 G1期細(xì)胞顯著高于其余3組，而G2 M期細(xì)胞顯著低于其余三組，DNA含量顯著低于其余3組；R組p27kip1表達(dá)明顯強(qiáng)于其余3組. 結(jié)論： PF4對(duì)放射損傷的BMSC有保護(hù)作用，可能與抑制p27kip1表達(dá)有關(guān).
【關(guān)鍵詞】 骨髓基質(zhì)細(xì)胞；血小板因子4；輻射損傷；輻射防護(hù)；p27
0引言
造血功能障礙是放射損傷時(shí)最基本的病理變化. 此時(shí)的造血功能障礙涉及造血干/祖細(xì)胞和造血基質(zhì)細(xì)胞損傷. 骨髓基質(zhì)細(xì)胞(bone marrow stromal cell, BMSC)是造血誘導(dǎo)微環(huán)境(hematopoietic inductive microenvironment, HIM)的重要組成成分. 血小板第4因子(PF4)能可逆地抑制造血干細(xì)胞、祖細(xì)胞的生長，又是骨髓細(xì)胞調(diào)節(jié)的負(fù)調(diào)節(jié)因子,并因此而保護(hù)骨髓造血干祖細(xì)胞免受輻射和細(xì)胞毒劑的損傷［1，2］. 在本實(shí)驗(yàn)中我們首次以體外培養(yǎng)放射損傷小鼠的BMSC為基礎(chǔ)，觀察PF4對(duì)其的保護(hù)作用，進(jìn)一步探討PF4對(duì)放射損傷的保護(hù)機(jī)制.
1材料和方法
1.1材料
BABL/c健康雄性小鼠40只，體質(zhì)量22~24 g，第四軍醫(yī)大學(xué)實(shí)驗(yàn)動(dòng)物中心提供；RPMI1640培養(yǎng)基（Gibco）；PF4（Sigma）：用雙蒸水稀釋成工作液,置4℃冰箱保存?zhèn)溆?稀釋濃度為100 mg/L；特級(jí)小牛血清、馬血清（杭州，四季青）；胰酶（Sigma）；P27kip1, SP試劑盒及DAB顯色試劑盒（武漢博士德）.
1.2方法
60Co γ射線一次全身均勻照射,吸收劑量為5.0 Gy,吸收劑量率為 221.09 cGy/min，距離100 cm. 實(shí)驗(yàn)隨機(jī)分4組，每組10只小鼠： ① 單純放射組（單放組，R）： 5.0 Gy 60Co γ射線全身照射；②放射＋PF4組（R P）： 放射前26和20 h給予PF4 ip，50 μg/kg，照射方式和劑量同①組；③正常＋PF4組（N＋P）：PF4注射時(shí)間及劑量同②組，但不照射；④正常對(duì)照組（N）： 不照射及PF4注射. BMSC原代培養(yǎng)參照Dexter方法［3］： BAB
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