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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 effects of knockdown of microRNA-7 (miR-7) on mouse splenic T lymphocytes in vitro and its significance. The spleen T lymphocytes were isolated from wild-type (WT) mice and the expression of miR-7 was detected by Real-time PCR at different time points (0 h, 24 h and 48 h) after stimulation with CD3 and CD28 antibodies The spleen T lymphocytes of miR-7KD mice were stimulated with Con A, CD3 and CD28 antibodies, and the proliferation rate of cells was detected by CCK8. The levels of IL-12, IL-4 and IL-6 in splenic T lymphocytes of miR-7KD mice were detected by Real- The changes of CD4 + T and CD8 + T cells and the expressions of CD44, CD62L, IL-4 and IFN-γ in CD4 + T cells were detected by FACS. The results showed that the expression of miR-7 was significantly up-regulated in splenic T lymphocytes of WT mice (P <0.05). Compared with WT mice, the spleens of miR-7KD mice under the action of Con A, CD3 and CD28 (P <0.05). The levels of IL-12, IL-4, IL-6, TNF-α and IFN-γ in spleen T lymphocytes of miR-7KD mice were significantly increased While the expression of IL-10 was significantly down-regulated (P <0.05). The results of FACS showed that the proportion of CD4 + T cells was significantly increased (P <0.05), while the proportion of CD8 + T cells was not significantly changed The level of CD62L was significantly decreased, and the expressions of CD69, IL-4 and IFN-γ were significantly up-regulated (P <0.05). The results showed that miR-7 knockdown can significantly affect the function of mouse splenic T lymphocytes, the experimental follow-up to explore in depth its role in T lymphocyte function regulation provide experimental evidence.