同型半胱氨酸对培养的人单核细胞株THP-1表达巨噬细胞炎性蛋白-1α的影响

Effect of homocysteine on the expression of macrophage inflammatory p rotein-1α in THP-1 monocytes

目的 :探讨同型半胱氨酸对人单核细胞株THP -1细胞表达巨噬细胞炎性蛋白 -1αmRNA和蛋白的影响。方法 :在体外培养的THP -1单核细胞培养基中加入终浓度为 0 .0 5mmol/L、0 .1mmol/L和 0 .2mmol/L的同型半胱氨酸 ,孵育 8h ;或加入终浓度 0 .1mmol/L的同型半胱氨酸 ,分别孵育 4、8、16h。用逆转录聚合酶链反应检测THP -1单核细胞巨噬细胞炎性蛋白 -1αmRNA ,并测序 ;用免疫细胞化学法检测巨噬细胞炎性蛋白 -1α蛋白的表达。结果 :对照组THP -1单核细胞表达较低水平的巨噬细胞炎性蛋白 -1αmRNA ,加同型半胱氨酸后巨噬细胞炎性蛋白 -1αmRNA表达随浓度增加而增强 ;在浓度为 0 .1mmol/L时 ,巨噬细胞炎性蛋白 -1αmRNA表达于 4h开始升高 ,8h后逐渐降低。反应产物真实性经测序反应证实。免疫细胞化学图像分析结果显示 ,各组细胞平均吸光度值随浓度和时间递增而增加 (P <0 .0 1)。结论 :同型半胱氨酸能诱导THP -1单核细胞表达巨噬细胞炎性蛋白 -1αmRNA和蛋白。

AIM: To investigate whether homocysteine (HCY) induce the expression of macrophage inflammatory protein-1α(MIP-1α)in cultured THP-1 monocytes. METHODS: After exposure of THP-1 monocytes to HCY at increasing conce ntrations (0.05,0.1 and 0.2 mmol/L) for 8 h, or at 0.1 mmol/L of HCY for differe nt incubation times (4, 8 and 16 h), the expressions of MIP-1α mRNA and protein were determi n ed by RT-PCR and immunocytochemistry, respectively. RESULTS: RT-PCR showed that t he expression of MIP-1α mRNA increased with the concentrations of HCY compared with the control group. Meanwhile, after the treatment of 0.1 mmol/L HCY to t he cells for different times, the MIP-1α mRNA expression increased at 4 h, peak ed at 8 h, and then decreased at 16 h. The authenticity of RT-PCR products was c onfirmed by DNA sequencing. Image analysis of Immunocytochemistry assay showed t he expression of MIP-1α protein in experimental groups increased in a dose- and time-dependent manner(P<0.01). CONCLUSIONS: HCY induced monocytes to express MIP-1α mRNA and protein.