Alpha-黑素细胞刺激素及其新型类似物对小鼠脑微血管内皮细胞产生组织因子的抑制作用

目的研究Alpha-黑素细胞刺激素(α-MSH)及其新型类似物(STY39)对原代小鼠脑微血管内皮细胞(MBMEC)在脂多糖(LPS)刺激下产生组织因子(TF)和组织因子途径抑制物(TFPI)的影响。方法选用雌性BALB/c小鼠,纯化并原代培养MBMEC,培养至5~7d,采用免疫荧光法检测Ⅷ因子相关抗原,鉴定MBMEC模型。将MBMEC分为PBS对照组,LPS刺激组,LPS刺激后1、2、3 h加入10-7mol/L的α-MSH或STY39组(LPS+α-MSH,LPS+STY39),共8组,每组4孔。分别在LPS刺激后6h和8h收集细胞培养上清液和细胞,应用酶联免疫吸附法检测细胞上清液的TF和TFPI浓度,RT-PCR检测细胞TF mRNA表达水平。结果 (1)LPS可诱导MBMEC产生TF和TFPI蛋白,细胞培养上清液中TF水平于6 h达到高峰,TFPI水平于8 h达到高峰。(2)LPS刺激MBMEC后1、2、3 h给予10-7mol/L的α-MSH或STY39,均可显著降低细胞上清液中TF蛋白含量(P0.01),尤其在LPS刺激后1 h给予α-MSH或STY39的效果最显著(P0.05),STY39降低TF含量的作用较α-MSH明显(P0.05);但α-MSH和STY39对LPS诱导MBMEC产生TFPI均没有显著的上调作用。(3)在LPS刺激后不同时间点给予10-7mol/L的α-MSH或STY39,均可显著下调MBMEC TF mRNA的表达水平(P0.01),其中1 h时间点作用最显著(P0.05),但α-MSH与STY39的作用差异无统计学意义。结论α-MSH和STY39均能抑制LPS诱导原代MBMEC产生TF蛋白和表达TF mRNA,且LPS刺激1 h后给药效果较好;STY39对MBMEC产生TF蛋白的抑制作用优于α-MSH。

Inhibitory effect of alpha-melanocyte stimulating hormone and its novel analogue on the production of tissue factor in mouse brain microvascular endothelial cells

Objective To study the effect of alpha-melanocyte stimulating hormone （α-MSH） and its novel analogue （ STY39 ） on the production of tissue factor （ TF ） and tissue factor pathway inhibitor （TFPI） stimulated by lipopolysaccharide （LPS） in primary mouse brain microvascular endothelial cells （MBMECs）. Methods Female BALB/c mice were selected,purified and primarily cultured for 5 to 7 days. Immunofluorescence assay was use to detect the Ⅷ factor related antigen and identify the MBMEC model. The MBMECs were divided into eight groups：PBS control group, LPS stimulation group, after LPS stimulation 1,2,and 3 h adding 10 -7 mol/Lα-MSH groups or STY39 group （LPS＋α-MSH,LPS＋STY39） （ n=4 wholes in each group） . The cell culture supernatant and cells were collected at 6 and 8 h after LPS stimulation. An enzyme-linked immunosorbent assay was used to detect the concentrations of TF and TFPI＆amp;nbsp;in cell supernatant. RT-PCR was used to detect the expression levels of TF mRNA. Results （1） LPS could induce MBMEC to produce TF and TFPI proteins. The level of TF in the cell culture supernatant reached the peak at 6 h,and the level of TFPI reached the peak at 8 h. （2） At 1,2,and 3 h after LPS stimulating MBMEC,10 -7mol/L α-MSH or STY39 were given. They could significantly decrease the TF protein content in the cell supernatant （P〈0. 01）,especially the effects of giving α-MSH or STY39 were most significant at 1 h after LPS stimulation （P〈0. 05）. The effect of STY39 for decreasing TF content was more significant than that of α-MSH （P〈0. 05）;however,α-MSH and STY39 did not have significant up-regulating effects for LPS inducing MBMEC to produce TFPI. （3） After LPS stimulation,10 -7 mol/Lα-MSH or STY39 were given at different time points. They significantly down-regulated the expression level of MBMEC TF mRNA （P〈0. 01）. The effect was most significant at 1 h time point （P〈0. 05）,but there was no significant difference in the effects betweenα-M