LPS与巨噬细胞中htra2的表达及相关性研究

Study of Expression and Relevance of htra2 in Macrophage and LPS

通过LPS(lipopolysaccharide)分别刺激树突状细胞和巨噬细胞,发现巨噬细胞中HtrA丝氨酸蛋白酶2(HtrA serine peptidase 2,htra2)基因的表达随着刺激时间的延长呈现下调的变化趋势,提示巨噬细胞中的htra2受到LPS的调控。为了进一步证实LPS刺激的特异性,实验建立VSV(vesicular stomatitis virus)病毒感染巨噬细胞模型,采用荧光定量PCR,免疫荧光方法分别从htra2的蛋白水平和定位上研究htra2的变化与LPS刺激巨噬细胞产生效应的相关性;构建了htra2基因过表达的质粒pKH3-htra2-HA,采用ELISA,Western Blot及荧光定量PCR等方法,检测过表达htra2对巨噬细胞产生和分泌炎症因子IL-6(Interleukin-6)及IL-1b(Interleukin-1b)的影响。实验结果表明:htra2仅在LPS刺激巨噬细胞的体系中受到调控,且过表达的htra2会上调巨噬细胞IL-1b和IL-6的表达水平。

This study stimulates dendritic eel[ and maerophage with LPS（lipopolysaccharide） and finds that the expression of HtrA serine peptidase 2（htra2） gene presents a variation trend of down-regulation with the extension of stimulation time, indicating that htra2 in macrophage is regulated and controlled by LPS. To further demonstrate the specificity of LPS stimulation, the experiment establishes VSV（Vesicu- lar Stomatitis Virus） virus infected macrophage model and studies the correlation between htra2 variation and effect of LPS stimulation on macrophage respectively from the protein level and positioning of htra2 with fluorescent quantitation PCR and immunoflurocence methods~ establishes plasmid pKH3-htra2-HA of htra2 gene overexpression and detects the influence of overexpressed htra2 on macrophage＇s production and secretion of inflammatory factors IL-6 （Interleukin-6） and IL-lb （Interleukin-lb） with ELISA, Western Blot and fluorescent quantitation PCR. The experimental result shows that htra2 is only regulated and controlled in the system of LPS stimulating macrophage and overexpressed htra2 will cause up-regulation of expression level of macrophage IL-lb and IL-6.