CAJ | 학술논문

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5~7 d,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6 h和8 h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(P<0.01),尤其在 LPS刺激后1 h给予α-MSH 或 STY39的效果最显著(P <0.05), STY39降低TF含量的作用较α-MSH明显(P<0.05)；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(P<0.01),其中1 h时间点作用最显著(P<0.05),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。
목적：연구Alpha-흑소세포자격소(α-MSH)급기신형유사물(STY39)대원대소서뇌미혈관내피세포( MBMEC)재지다당( LPS)자격하산생조직인자( TF)화조직인자도경억제물( TFPI)적영향。방법선용자성BALB/c소서,순화병원대배양MBMEC,배양지5~7 d,채용면역형광법검측Ⅷ인자상관항원,감정MBMEC모형。장MBMEC분위PBS대조조,LPS자격조,LPS자격후1、2、3 h가입10-7mol/L적α-MSH혹STY39조(LPS+α-MSH,LPS+STY39),공8조,매조4공。분별재LPS자격후6 h화8 h수집세포배양상청액화세포,응용매련면역흡부법검측세포상청액적TF화TFPI농도,RT-PCR검측세포TF mRNA표체수평。결과(1) LPS 가유도 MBMEC 산생 TF 화TFPI단백,세포배양상청액중TF수평우6 h체도고봉, TFPI수평우8 h체도고봉。(2) LPS자격 MBMEC후1、2、3 h급여10-7mol/L적α-MSH혹 STY39,균가현저강저세포상청액중 TF단백함량(P<0.01),우기재 LPS자격후1 h급여α-MSH 혹 STY39적효과최현저(P <0.05), STY39강저TF함량적작용교α-MSH명현(P<0.05)；단α-MSH화STY39대LPS유도MBMEC산생TFPI균몰유현저적상조작용。(3)재LPS자격후불동시간점급여10-7mol/L적α-MSH 혹STY39,균가현저하조MBMEC TF mRNA적표체수평(P<0.01),기중1 h시간점작용최현저(P<0.05),단α-MSH여STY39적작용차이무통계학의의。결론α-MSH화 STY39균능억제LPS유도원대 MBMEC 산생 TF 단백화표체 TF mRNA,차 LPS 자격1 h 후급약효과교호；STY39대MBMEC산생TF단백적억제작용우우α-MSH。
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 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-MSH and STY39. Conclusion Bothα-MSH and STY39 can suppress LPS-induced primary MBMEC to produce TF protein and express TF mRNA,and the effect of administration is better after 1 h LPS stimulation. The suppressive effect of STY39 on the production of TF protein is superior toα-MSH.