同型半胱氨酸诱导人单核细胞表达RANTES及其受体CCR1

Homocysteine induced human monocytes to express RANTES and CCR1

目的观察不同浓度同型半胱氨酸对人单核细胞RANTES因子、趋化因子受体CCR1及核因κBP65表达的影响,及NF-κB在人单核细胞表达RANTES及其受体CCR1中所起的作用。方法将生长状态良好的THP-1细胞随机分为对照组、Hcy实验组及二硫代氨基甲酸吡咯烷(PDTC)预处理组。对照组用不含Hcy的无血清培养基、Hcy实验组分别用不同Hcy终浓度的无血清培养基孵育8 h,PDTC预处理组在PDTC终浓度100μmol/L的无血清培养基中孵育20 min后,再分别在含不同Hcy终浓度的无血清培养基中孵育8h。采用RT-PCR及免疫细胞化学方法检测各组细胞中RANTES及其受体CCR1 mRNA及蛋白表达情况,用Western-blotting检测NF-κB P65蛋白的表达。结果 RANTES和CCR1的mRNA及蛋白在Hcy实验组的表达均随Hcy浓度增加而增强,均显著高于正常对照组和PDTC预处理组中Hcy浓度相对应组;NF-κB P65蛋白在Hcy实验组各浓度组的表达随Hcy浓度增加而增强,均显著高于对照组和PDTC预处理组Hcy浓度相对应组。结论 Hcy呈剂量依赖性诱导THP-1细胞中RANTES及其受体CCR1 mRNA和蛋白、NF-κB P65蛋白的表达。Hcy可能通过NF-κB途径诱导人单核细胞表达趋化因子RANTES及其受体CCR1而在动脉粥样硬化的形成中起作用。

[Objectives ] To investigate the effect of homocystein at different concentrations on the expressions of RANTES, the ehemoattractant cytokine receptor-1 and nuclear factor-KB p65 in human monocytes, the role of NF-KB in the expressions of RANTES and CCR1. [Methods] THP-1 cells in good condition were divided into three groups including the control group, the group only treated with homocysteine, and pyrrolidine dithiocarbamate pretreatment group at random. The cells of the control group were cultured only with the media without serum. The cells in the group only treated with homocysteine were stimulated with ho- mocysteine at different concentrations in serum-free conditions, respectively. The two groups mentioned above were incubated for 8 hours, respectively. The group pretreated with PDTC was incubated with PDTC at a concentration of 100 Izmol/L for 20 minutes at first, then exposed to homocysteine at different concentration in serum-free conditions for 8 hours. The expressions of RANTES and CCR1 mRNA and protein were detected with RT-PCR and immunocytochemistry. The expression of NF-KBp65 protein was detected with West- ern-blotting. [Results] The expressions of RANTES and CCR1 mRNA and protein were significantly increased by Hcy in a dose-dependent manner, which were dramatically higher than those in the normal control group. Pretreatment with PDTC significantly decreased expressions of RANTES and CCR1 mRNA and protein in the cells treated with Hcy, respectively. The expressions of NF-KB p65 protein in the group treated only with Hcy group dramatically increased in a dose-dependent manner, which was much higher than that of the nor- mal control group. Pretreatment with PDTC significantly decreased expression of NF-KB p65 protein in the cells treated with Hcy. [Conclusions] Homocysteine induced the expressions of RANTES and CCR1 mRNA and protein and NF-KB p65 protein in THP-1 cells in. a dose-dependent manner. Homocysteine may play a key role in atherogenesis by accelerating the expression of RANTES and CCR1 in human monocytes via NF-KB pathway.