【作者】 王丹丹；

【导师】 胡秀芬； 王宏伟；

【作者基本信息】 华中科技大学 ， 儿科学， 2006， 硕士

【摘要】 目的观察川崎病急性血清和丙种球蛋白对血管内皮细胞白三烯B4受体1(BLT1,leukotriene B4 receptor 1)基因的表达及内皮细胞产生白三烯B4(LTB4,leukotriene B4)的影响,并观察不同刺激后的内皮细胞条件培养基对小鼠腹腔巨噬细胞表达白三烯B4受体2(BLT2,leukotriene B4 receptor 2)的影响,以了解LTB4-BLT通路在川崎病血管损伤过程中的作用以及丙种球蛋白治疗川崎病可能的作用机制。方法1.内皮细胞培养和实验分组:人脐静脉内皮细胞(ECV304)按以下设计分组,A正常血清组:RPMI1640培养基+10%健康儿童血清;B川崎病血清组:RPMI1640培养基+10%川崎病急性期血清;C丙种球蛋白组:RPMI1640培养基+10%川崎病急性期血清+丙种球蛋白(20mg/ml)。留各组细胞刺激液,将细胞分别培养24,48小时后,用TRIzol溶解细胞,-70℃冻存,并收集细胞培养上清液作为条件培养基备用。2.采用逆转录聚合酶链式反应(RT-PCR)法对内皮细胞BLT1mRNA进行扩增,产物琼脂糖凝胶中电泳后,用凝胶成像分析系统测定电泳条带密度值,以β-actin为内参照,计算BLT1基因mRNA的相对含量,结果以BLT1与β-actin条带密度的比值表示。3.各组内皮细胞培养48小时后,采用酶联免疫吸附试验(ELISA)检测培养液培养前后的LTB4浓度。4.分离健康Balb/c小鼠腹腔巨噬细胞,将细胞分为正常血清条件培养组,川崎病血清条件培养组和丙种球蛋白条件培养组。在各组细胞培养瓶中加入上述各组内皮细胞条件培养基,继续培养24小时后,用0.25%胰蛋白酶溶液消化细胞备检。5.采用流式细胞技术将消化下来的各组巨噬细胞用间接免疫荧光标记法检测细胞BLT2的表达。结果1.川崎病血清组内皮细胞BLT1的表达水平在培养的第24、48小时分别为(54.23±6.25)%、(67.17±3.49)%,显著高于正常血清组(25.63±3.62)%和丙种球蛋白组(32.30±6.89)%(p<0.05)。正常血清组和丙种球蛋白组BLT1的表达水平无显著性差异(p>0.05)。2. LTB4的浓度在川崎病血清刺激48小时后为(595.6±46.4)pg/ml显著高于刺激前的浓度(106.3±45.3)pg/ml(p<0.05);而在用含治疗量丙种球蛋白培养液培养的上清中LTB4浓度上调不明显。3.川崎病血清刺激后的内皮细胞条件培养基能够使巨噬细胞BLT2表达上调[(33.16±4.08)% VS(12.43±3.71)%](p<0.05);而加入丙种球蛋白后可抑制上述BLT2的改变。结论LTB4-BLT通路参与川崎病血管内皮损伤过程并在川崎病的血管损伤中发挥着重要作用,丙种球蛋白可能通过阻断LTB4-BLT通路而起到治疗川崎病的作用。更多还原

【Abstract】 ObjectiveTo investigate the effect of the serum taken from patients with acute Kawasaki disease andγ-globulin on the expressions of BLT1 at the level of mRNA and LTB4 production in endothelial cell. Meanwhile, the effects of the endothelial cell conditioned culture media (ECM) on the expression of BLT2 in macrophage were observed. This study aimed at exploring whether LTB4-BLT pathway is involved in endothelial damage in Kawasaki disease and the mechanism ofγ-globulin in the treatment of Kawasaki disease.Methods1. Groups of cultured endothelial cell (ECV304): A. normal serum group: RPMI1640 culture medium+10% blood serum of healthy children. B. blood serum of KD group: RPMI1640 culture medium+10% blood serum of KD. C.γ-globulin group: RPMI1640 culture medium+10% blood serum of KD+γ-globulin (20mg/ml). The cells in each group were cultured for 24h and 48h, then dissolved with TRIzol. The culture medium in each group was collected before and after cultured.2. The mRNA levels of BLT1 were amplified by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). Products were segregated by agarose gel electrophoresis. Gel imaging analysis device was used to analyze the results of the agarose gel electrophoresis. The results were indicated by optical density of the BLT1/β-actin.3. The concentration of LTB4 in the supernatant was determined by ELISA before and after cultivation of 48h.4. Balb/c mouse macrophages were separated and cultured in conditioned media containing the serum of KD orγ-globulin. The macrophages were cultured for 24h and prepared by 0.25% trypsin in each ECM.5. The expression of BLT2 in the macrophage was determined by flow cytometry.Results1. The expression of BLT1 in the ECV304 was upregulated 24h and 48h after the stimulation by the serum of Kawasaki disease(p<0.05), but was not significantly upregulated in either normal serum group orγ-globulin group.2. The concentration of LTB4 in the endothelial cell culture was significantly increased by the serum of patients with KD(p<0.05 VS normal control), but not by normal conditioned media orγ-globulin conditioned media.3. The expression of BLT2 in the macrophage was enhanced significantly(p<0.05 VS normal control)by the serum of patients with KD. But such effect was abolished by addition ofγ-globulin.ConclusionsIt is assumed that LTB4-BLT pathway is involved in vascular damage in Kawasaki disease, and blocking the LTB4-BLT pathway may be one of the mechanism ofγ-globulin to treat Kawasaki disease.更多还原