microRNA-7基因敲减对小鼠CD4^+SP细胞胸腺发育的影响

Influence of microRNA-7 knock down on development of CD4^+SP cells in murinethymus

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CD62L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。

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.