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
目的：探讨脓毒症单核细胞表达ILT4的生物学行为和效应，及对于预后的影响。方法：选取BALB/c、BALB/c ILT4–/–雄性小鼠，CLP复制脓毒症模型。用流式细胞仪定量检测CLP术后24h单核细胞ILT4、MHC-Ⅱ表达水平；用ELISA法检测各组0、6、1...目的：探讨脓毒症单核细胞表达ILT4的生物学行为和效应，及对于预后的影响。方法：选取BALB/c、BALB/c ILT4–/–雄性小鼠，CLP复制脓毒症模型。用流式细胞仪定量检测CLP术后24h单核细胞ILT4、MHC-Ⅱ表达水平；用ELISA法检测各组0、6、12、24h血清IL-6、TNF-α浓度；并观察168h内生存预后。 结果：CLP术后24小时脓毒症小鼠外周单核细胞高度表达ILT4分子 （193.50 ± 52.54 vs. 1292.00 ± 143.70, p 〈 0.05） ；较WT组ILT4–/–小鼠外周单核细胞表达MHC-Ⅱ比率明显增高（24.25± 6.76% vs. 49.38 ±5.66%, p 〈 0.05） 。CLP术后24h血清IL-6显著增高（54.25± 20.04 vs. 470.75±88.03, p 〈 0.05），ILT4敲除后很大程度的抑制这一趋势（470.75±88.03 vs. 241.25 ± 45.10, p 〈 0.05）；但对TNF-α表达无显著性干扰（53.13 ±5.49 vs. 50.88 ±6.38, p〉 0.05）。且ILT4–/–小鼠CLP术后生存率WT明显增加（p 〈 0.05）。结论：脓毒症时单核细胞高表达ILT4，与高IL-6水平及低MHC-Ⅱ表达率相关，导致死亡率增加。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.
文摘目的：探讨脓毒症单核细胞表达ILT4的生物学行为和效应，及对于预后的影响。方法：选取BALB/c、BALB/c ILT4–/–雄性小鼠，CLP复制脓毒症模型。用流式细胞仪定量检测CLP术后24h单核细胞ILT4、MHC-Ⅱ表达水平；用ELISA法检测各组0、6、12、24h血清IL-6、TNF-α浓度；并观察168h内生存预后。 结果：CLP术后24小时脓毒症小鼠外周单核细胞高度表达ILT4分子 （193.50 ± 52.54 vs. 1292.00 ± 143.70, p 〈 0.05） ；较WT组ILT4–/–小鼠外周单核细胞表达MHC-Ⅱ比率明显增高（24.25± 6.76% vs. 49.38 ±5.66%, p 〈 0.05） 。CLP术后24h血清IL-6显著增高（54.25± 20.04 vs. 470.75±88.03, p 〈 0.05），ILT4敲除后很大程度的抑制这一趋势（470.75±88.03 vs. 241.25 ± 45.10, p 〈 0.05）；但对TNF-α表达无显著性干扰（53.13 ±5.49 vs. 50.88 ±6.38, p〉 0.05）。且ILT4–/–小鼠CLP术后生存率WT明显增加（p 〈 0.05）。结论：脓毒症时单核细胞高表达ILT4，与高IL-6水平及低MHC-Ⅱ表达率相关，导致死亡率增加。
