Xu Xiaoli, Zou Ping, Chen Lijuan, Jin Guannan, Zhou Hao
Huazhong University of Science and Technology, Wuhan, China.
J Huazhong Univ Sci Technolog Med Sci. 2010 Oct;30(5):594-8. doi: 10.1007/s11596-010-0548-8. Epub 2010 Nov 10.
This study examined the effect of IL-10 on immunoglobulin-like transcript (ILT4) expression of human monocytic leukemic cell line THP-1, especially the role of the ILT4 promoter activity. ILT4 promoter area was amplified by PCR, and was cloned into the eukaryotic expressing vector pGL3-Basic. The pGL3-ILTP obtained was tested by double endonuclease digestion and sequencing. Then, the recombinant plasmid was transfected into THP-1 cells by using lipofectamine. After culture with IL-10 for 12 h, the mRNA extracted from THP-1 cells was detected by RT-PCR and the protein was detected by FACS. The dual-luciferase reporter assay system was employed to detect the activity of ILT4 promoter with or without IL-10. The results showed that the activity of pGL3-ILTP was significantly increased and was more than ten times that of pGL3-Basic cells. After culture with IL-10 for 12 h, the expression of ILT4 protein and its mean fluorescence intensity (MFI) were increased. Moreover, the mRNA was remarkably higher than that of the control group. Dual-luciferase reporter assay revealed that ILT4 promoter was much more activated after being treated with IL-10. We were led to conclude that pGL3-ILTP containing ILT4 promoter was constructed successfully. The expression of ILT4 could be up-regulated by IL-10 both at the transcriptional and translational level. Furthermore, ILT4 promoter could be much more active after addition of IL-10. This study suggests that IL-10 up-regulates ILT4 expression on monocytes via increasing ILT4 gene promoter activity, which may have implication for inducing transplantation tolerance in clinical practice.
本研究检测了白细胞介素-10（IL-10）对人单核细胞白血病细胞系THP-1免疫球蛋白样转录物（ILT4）表达的影响，尤其是ILT4启动子活性的作用。通过聚合酶链反应（PCR）扩增ILT4启动子区域，并将其克隆到真核表达载体pGL3-Basic中。对获得的pGL3-ILTP进行双酶切和测序检测。然后，使用脂质体转染试剂将重组质粒转染到THP-1细胞中。用IL-10培养12小时后，通过逆转录-聚合酶链反应（RT-PCR）检测从THP-1细胞中提取的mRNA，并通过荧光激活细胞分选术（FACS）检测蛋白质。采用双荧光素酶报告基因检测系统检测有无IL-10时ILT4启动子的活性。结果显示，pGL3-ILTP的活性显著增加，是pGL3-Basic细胞活性的十多倍。用IL-10培养12小时后，ILT4蛋白的表达及其平均荧光强度（MFI）增加。此外，mRNA明显高于对照组。双荧光素酶报告基因检测显示，用IL-10处理后ILT4启动子的激活程度更高。我们得出结论，成功构建了含ILT4启动子的pGL3-ILTP。IL-10可在转录和翻译水平上调ILT4的表达。此外，添加IL-10后ILT4启动子的活性更高。本研究表明，IL-10通过增加ILT4基因启动子活性上调单核细胞上ILT4的表达，这可能对临床实践中诱导移植耐受具有重要意义。
