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 amplifi...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.展开更多
基金supported by grants from National Natural Sciences Foundation of China (No.30571755)Specialized Research Fund for the Doctoral Program of Higher Education (No.200804871175)
文摘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.
