微小RNA-7敲减对小鼠脾脏中T淋巴细胞体外功能的影响

The effect of miRNA-7 knock down on the function of T lymphocyte of spleen in vitro

观察微小RNA-7(microRNA-7,miR-7)敲减(Knock down,KD)对小鼠脾脏T淋巴细胞体外功能的影响并探讨其意义。常规分离野生型(wild type,WT)小鼠脾脏T淋巴细胞,经CD3和CD28抗体刺激后,Real-time PCR检测不同时间点(0h;24h;48h)细胞中miR-7的表达变化;进一步用Con A、CD3和CD28抗体刺激miR-7KD小鼠脾脏T淋巴细胞,CCK8检测细胞增殖率;Real-time PCR检测miR-7KD小鼠脾脏T淋巴细胞IL-12、IL-4、IL-6、TNF-α、IFN-γ和IL-10表达的变化;FACS检测CD4+T和CD8+T细胞的数量变化及CD4+T细胞膜分子CD44、CD62L和IL-4、IFN-γ的表达变化。结果显示,WT小鼠脾脏T淋巴细胞活化后,miR-7的表达水平显著上调(P<0.05);与WT小鼠相比,在Con A、CD3和CD28抗体作用下miR-7KD小鼠脾脏T淋巴细胞增殖明显增加(P<0.05);miR-7KD小鼠脾脏T淋巴细胞IL-12、IL-4、IL-6、TNF-α和IFN-γ水平均明显上调(P<0.05),而IL-10表达显著下调(P<0.05);FACS检测结果显示CD4+T细胞比例明显上调(P<0.05),而CD8+T细胞的比例变化不显著(P>0.05);CD4+T细胞膜分子CD62L水平显著下降,CD69及IL-4、IFN-γ的表达水平均显著上调(P<0.05)。结果表明,miR-7敲减以后可显著影响小鼠脾脏T淋巴细胞的功能,本实验为后续深入探讨其在T淋巴细胞功能调控中的作用提供实验依据。

To observe the effect of miRNA-7 knock down on the function of spleen T lymphocytes in vitro, and preliminary ex- plore its possible significance ,spleen T lymphoeytes derived from wild-type （WT） mice were stimulated with anti-CD3/CD28 antibodies in vitro. Then, the relative expression of miR-7 at different time points （0 h, 24 h, 48 h） was examined by Real- time PCR. Furthermore, spleen T lymphocytes derived from miR-7 knock down （KD） mice were stimulated with Con A and anti-CD3/CD28 antibodies respectively, the proliferation of cells was examined by CCK-8 assay, the relative expressions o{ IL- l2, IL-4, IL-6, TNF-a, IFN-y and IL-10 in the T lymphocytes were determined by Real-time PCR. Finally, the change of pro-portion of both the CD4＋ T cells and CD8＋ TCells in the total spleen T lymphocytes were analyzed by FACS. Moreover, the ex- pression levels of CD44 and CD62L molecules as well as IL-4 and IFN-γ in CD4＋ T cells were analyzed by FACS. The results showed that the relative expression level of miR-7 in activated spleen T lymphocytes derived from WT mice was significantly upregulated （P〈0.05） and the proliferation level of spleen T lymphocytes stimulated with Con A and anti-CD3/CD28 antibod- ies was significantly increased in miR-7 KD mice compared with that in WT mice （P〈0.05）. RT-PCR results showed that the expressions of IL-12, IL-4, IL-6, TNF-a and IFN-γ were also markedly upregulated in miR-7 KD mice （P〈0.05）, while IL- 10 expression was significantly decreased （P〈0.05）. Finally, FACS analysis indicated that the proportion of CD4＋ T cells in the spleen T lymphocytes was significantly increased in miR-7 KD mice compared with that in WT mice （P〈0.05）, whereas there was no difference in the proportion of CDS＋T cells between the two groups （P〉 0.05）. Meanwhile, we also found that the expression of CD62L molecules was significantly declined, but the expressions of CD69 molecule and IL-4, IFN-γ were sig- nificantly upregulated （P〈0.05） in miR-7 KD mice. Our results thus indicate that knock down of miR-7 can significantly af- fect the function of spleen T lymphocytes in vitro, which provides an important foundation for successive research work on ex- ploring the functional role of miR-7 in T lymphoeytes.