microRNA-7基因敲减对小鼠CD4＋SP细胞胸腺发育的影响

摘要

Objective:To investigate the influence of miR-7 knock down on the development of CD 4+SP cells in the thymus in mice,and preliminary explore its possible mechanism.Methods:The changes of volume ,weight and total cell counts of thymus in miR-7 knock down (miR-7KD) mice were observed compared with Wild-type(WT)mice;the pathological changes of thymus were observed by HE staining.FACS analysis was performed on the proportion ,as well as the expression level of CD44 and CD62L,of thymus CD4+single positive (SP) cells.Meanwhile,the proliferation percentage of CD4+SP cells was measured by Ki-67 staining.The apoptosis percentage of CD4+SP cells was analyzed by FACS.The changes on the transduction of ERK 1/2 pathways were determined by Western blot.Results:Compared with WT mice ,the size,weight and total cell number of thymus were marked reduced in miR-7KD mice( P<0.05 );moreover ,pathological change also was presented.The proportion and total cell number of thymus CD 4+SP cells were marked decreased ( P<0.05 ).Furthermore ,the expression level of CD 44 and proliferation percentage ,as well as apoptosis percentage ,of CD4+SP cells were obviously increased (P<0.05),however,the expression level of CD62L of CD4+SP cells were decreased (P<0.05). Finally,the level of total ERK1/2 and phosphor-ERK1/2 was decreased obviously ( P<0.05 ).Conclusion: miR-7 knock down can affect the development of CD 4+SP cells in the thymus , which might be closely related to the cell activation state and altered the transduction of ERK1/2 pathways.%目的：研究microRNA-7（ miRNA-7，miR-7）基因敲减对小鼠CD4＋SP细胞胸腺发育的影响。方法：检测miR-7基因敲减（miR-7 knock down，miR-7KD）和野生型（Wild-type，WT）小鼠胸腺重量及细胞总数变化；HE染色观察miR-7KD小鼠胸腺的形态学结构改变；FACS检测胸腺CD4＋单阳性（Single positive，SP）细胞的比例并计算细胞总数，同时检测CD4＋SP细胞CD44及CD62L表达变化；核抗原Ki-67染色法检测CD4＋SP细胞的增殖情况；FACS检测CD4＋SP细胞的凋亡变化；Western blot 技术检测胸腺中总ERK1／2和磷酸化ERK1／2表达水平变化。结果：与WT小鼠相比，miR-7KD小鼠胸腺体积、重量以及细胞总数均显著减少，且形态学结构发生改变（ P＜0.05）；FACS结果显示，miR-7KD小鼠胸腺CD4＋SP细胞比例明显降低，且细胞总数减少（ P＜0.05）。此外，CD4＋SP细胞的CD44表达水平显著增加，而CD62 L表达水平明显减少（ P＜0.05）；同时，CD4＋SP细胞增殖及凋亡比例均显著增加（P＜0.01）；最后，miR-7KD小鼠胸腺中ERK1／2以及磷酸化ERK1／2的表达均明显下调（ P＜0.05）。结论：miR-7基因敲减后可显著影响CD4＋SP细胞的胸腺发育，这可能与细胞活化水平及ERK1／2信号通路改变有关。