低浓度叔丁基过氧化氢对小鼠胚肺成纤维细胞L929增殖作用的影响

目的通过不同浓度的叔丁基过氧化氢（Tert-Butyl bydroperoxide，TBHP）作用于培养的小鼠胚肺成纤维细胞系L929细胞，观察低浓度的TBHP对细胞生长、增殖的影响。方法按TBHP的终浓度从低到高分别为：空白组（0μmol／L）、低浓度组（1μmol／L）、中浓度组（10μmol／L）和高浓度组（40μmol／L）分成四组。通过破壁灵芝孢子粉水提液（2．0s／L）作用L929细胞系24h，使细胞同步化，分别加入相应浓度的TBHP溶液。用MTT比色法检测细胞活力，并以溴脱氧尿嘧啶核苷（bromode-oxyuridine，BrdU）的免疫荧光染色法观察各组细胞的增殖变化，同时在相差显微镜下观察细胞的形态学变化。结果镜下观察发现，TBHP1μmol／L组细胞密度高于空白组和中、高浓度组，且BrdU阳性细胞数目最多，细胞生长旺盛；MTT结果显示TBHP1μmol／L组OD值最高，其次为空白组和中浓度组，高浓度组最低（P〈0．01），BrdU阳性荧光显色主要在刚刚分裂完成的细胞，呈现“镜像性”改变。结论低浓度TBHP对体外培养L929细胞的增殖、生长具有一定促进作用，BrdU阳性荧光显色细胞的“镜像性”改变是反映细胞增殖活性的有效判定指标。

Low Concentration TBHP Promotes the Proliferation of L929 Cells

Objective To investigate the growth and proliferation of the mouse embryonic lung fibroblast L929 cells after being treated with different concentration tert-butyl bydroperoxide（TBHP）. Methods L929 cells synchronized by Sporoderm-broken Ganoderma Lucidum Spore powder（ 2 g/L）were cultured in cell culture medium containing final concentration TBHP of 0, 1, 10 and 40 μmol/L. The cell synchronization was detected by flow-cytometry. The viability of L929 cells was measured by MTT assay, the proliferation of the cells was detected by fluorescence immunostaining of bromodeoxyuridine（BrdU）. Meanwhile, the growth and morphological changes of L929 cells were observed by inverted phase contrast microscope. Results Compared with three control groups, the number of positive BrdU stained cells in low concentration group was much more than that in any other groups under the same scale of field of vision. The cells in low concentration group（ TB- HP= 1μmol/L） showed a higher OD value（0.354 ±0.007） than that in blank group（0.319 ±0.016）, middle concentration group（0.263 ±0.010） and high concentration group（ 0. 229 ± 0. 008）, implying the higher viability of cells in this group（ n =6, P≤0.01）, and the positive BrdU luorescent cells were mainly stained in cells what just finished cleavage emerging changes of ＂mirror image＂. Conclusion The low concentration TBHP can facilitate the proliferation and growth of L929 cells in vitro. The changes of ＂mirror image＂ of positive BrdU luorescent cells are effective index for judging activity of cell proliferation.