逆转录病毒感染小鼠原代巨噬细胞的方法

巨噬细胞在调节免疫反应中发挥着重要作用,但是其难以转染的特点妨碍了对巨噬细胞调节免疫反应的分子机制进行深入的研究。利用HEK293T细胞包装携带GFP标记的逆转录病毒载体,分别感染处于不同分化阶段的巨噬细胞,通过检测细胞的生长状况和GFP阳性细胞比例,探究感染时间对病毒感染效率的影响。结果表明,在巨噬细胞分化的早期阶段感染重组的逆转录病毒效率最高,并且对细胞的分化和增殖无明显影响。接下来,利用此方法研究了MKP5对I型干扰素表达的调节作用。通过构建和包装表达MKP5的重组逆转录病毒,在巨噬细胞分化后的第一天进行感染,然后进行Western杂交和实时定量PCR分析。采用这一感染条件,不仅检测到大量外源MKP5的表达,并且发现过表达MKP5能显著抑制LPS诱导的IFN表达。因此在巨噬细胞分化早期感染逆转录病毒,可以有效地表达外源目的蛋白,研究这些蛋白分子在巨噬细胞中调控免疫反应的功能,为今后的免疫学研究提供了一种行之有效的技术方法。

Methods for retrovirus infection on mouse primary macrophages

Despite playing crucial roles in the regulation of inflammation and homeostasis, macrophages are notoriously difficult to be transducted by recombinant viral vectors including lentiviruses. Here, we used GFP-expressing retrovirus produced by HEK293T cells to test the infection efficiency in macrophages at various differentiation stages. We found that infecting macrophages on day 1 after in vitro differentiation resulted in the highest transduction efficiency without affecting the morphology or the growth of these cells. Subsequently, we utilized this approach to study the expression and function of MKP5 in macrophages. Our results demonstrated that when efficiently overexpressed, MKP5 was able to inhibit LPS-induced IFN expression in macrophages. Together, a new method for efficiently overexpressing exogenous proteins in macrophages was reported, which would facilitate the study of molecular mechanisms on the regulation of macrophages in immune responses in the future.