摘要
目的:观察miRNA-126基因敲减(Knock down,KD)小鼠脾脏中免疫细胞组成比例变化并探讨其意义。方法:Realtime PCR探针法检测miR-126KD小鼠脾脏中miR-126表达水平,计算脾脏总细胞数;HE染色观察脾脏组织的病理学变化;流式细胞术(FACS)分别检测脾脏中DCs细胞、巨噬细胞、γδT细胞、NKT细胞,CD3^+T细胞和其亚群以及CD19^+B细胞的比例并计算细胞绝对数;免疫印迹法(Western blot,WB)检测脾脏组织中磷酸化NF-κB和磷酸化Akt的表达水平。结果:与野生型(WT)小鼠相比,miR-126KD脾组织中miR-126表达水平明显降低(P<0.05),细胞总数明显增加(P<0.05),且发生明显病理学改变;固有免疫细胞中NK细胞的比例和细胞绝对数显著增加(P<0.05),但巨噬细胞的比例显著降低(P<0.05);适应性免疫细胞中CD3^+T细胞和CD4^+T细胞的比例和绝对细胞数都显著增加(P<0.05),而CD19^+B细胞仅绝对数显著增加(P<0.05);最后miRNA-126KD小鼠脾脏组织的磷酸化NF-κB和磷酸化Akt水平明显增加(P<0.05)。结论:miRNA-126敲减小鼠脾脏中各免疫细胞亚群的组成发生明显改变,可能与NF-κB和Akt信号通路传递变化有关,为后续探讨miR-126在机体免疫应答中的作用提供了前期实验基础。
Objective： To detect the change of composition of immune cells in the spleen of miR-126 knockdown（ miR-126KD）mice and preliminarily explore its significance. Methods： The expression level of miR-126 in spleens of miR-126 KD mice was determined by Realtime PCR. And the total number of splenocytes was calculated. The pathologic morphology change of the spleen was observed by HE staining. And the changes on proportion of DCs,macrophages cells,γδ T cells and NK T cells,CD3^＋T cells and its subgroup,as well as CD19^＋B cells in spleens of miR-126 KD mice were analyzed by Flow cytometry and calculated respectively. The level of phosphorylated AKt and NF-κB was analyzed by Western blot assay. Results： Compared with those of WT mice,the expression level of miR-126 decreased obviously（ P〈0. 05） and the total number of cells increased obviously in spleen of miR-126 KD mice（ P〈0. 05）.Moreover,the pathologic morphology of miR-126 KD mice was significantly changed. The proportion and number of NK T cells in the inherent cells were significantly increased（ P〈0. 05）,but the proportion of Mφ cells were significantly decreased（ P〈0. 05）. Meanwhile,the proportion and number of CD3^＋T cells and CD4^＋T cells in the adaptive immune cells were significantly increased（ P〈0. 05）,while the total number of CD19~＋B cells were significantly decreased（ P〈0. 05）. Last,the level of phosphorylation Akt and NF-κB increased obviously in spleen of miRNA-126 knockdown mice. Conclusion： Change obviously on the composition of immune cells subsets in the spleen of miRNA-126 knockdown mice,which it may be related to the altered level of phosphorylated AKt and NF-κB and provides a preliminary experimental basis for the further exploring the roles of miR-126 in immune response.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2016年第4期460-464,共5页
Chinese Journal of Immunology
基金
国家自然科学基金(No.31370918)
教育部新世纪优秀人才计划(NCET-12-0661)资助
