学位论文4 - 图文

徐州医学院硕士论文

TLR4 Tris TNF-α TIR Toll-like receptor 4

Hydroxymethylaminomethane Tumor necrosis factor-α Toll-interleukin 1 receptor region Toll样受体4 三羟甲基氨基甲烷 肿瘤坏死因子α

Toll-白细胞介素1受体域

VIN

Vinorelbine

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长春瑞滨

徐州医学院硕士论文

Toll样受体4在长春瑞滨致静脉内皮损伤中

作用的初步研究

中 文 摘 要

目的 初步探讨Toll样受体4在长春瑞滨致静脉内皮损伤中的作用。 方法 1．Toll样受体4在长春瑞滨致小鼠静脉炎组织的表达及意义：取20只BALB/c小鼠，按随机数字表随机分为2组，每组10只。实验组按38 mg/kg尾静脉注射3.2 g/L长春瑞滨；对照组尾静脉注射相同体积的生理盐水。于给药后48 h颈椎脱臼处死小鼠，并取近心端距穿刺点1 cm处鼠尾组织，4%多聚甲醛中固定后以0.5 mol/L EDTA进行脱钙1周，常规石蜡包埋，切片，苏木精-伊红染色后显微镜下分析。免疫组织化学染色观察Toll样受体4在小鼠尾静脉血管内皮细胞的表达。2．Toll样受体4在长春瑞滨致人脐静脉内皮细胞损伤中的作用研究：取长势良好的HUVEC，接种于FN处理的24孔板或无菌培养瓶中，待细胞生长至融合度为90%-95%时，加入长春瑞滨浓度为0.05ug/ml 的EGM-2培养基，共同培养1h后，PBS洗涤，换入新鲜培养基继续培养0、6、12小时。RT-PCR以及荧光定量PCR检测TLR4的转录情况；Western Blot检测TLR4蛋白表达情况；免疫荧光检测NF-κB p65的核转位情况。

结果 1．注射后48 h，实验组小鼠尾静脉血管出现内皮细胞脱落、血栓形成和炎细胞浸润，损伤部位内皮细胞高表达Toll样受体4，对照组小鼠尾静脉血管无明显损伤表现，血管内皮细胞Toll样受体4阴性或弱表达，两组差异有统计学意义（P＜0.05）。2．0.05 ug/ml长春瑞滨干预1h后人脐静脉内皮细胞拉伸，延展，形态不规则，边界不清，排列紊乱，部分细胞悬浮脱落。洗去长春瑞滨换入新鲜培养基继续培养6h、12h后，细胞形态逐渐恢复正常；荧光定量PCR法检测各组细胞TLR4基因表达水平，结果显示：与空白对照组相比，各实验组TLR4表达量均升高，

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徐州医学院硕士论文

差异有统计学意义（P＜0.05），其中干预1h后升高幅度最大；Western Blot检测各组细胞TLR4蛋白表达水平，结果显示：与空白对照组相比，各实验组TLR4蛋白表达量均升高，差异有统计学意义（P＜0.05），随着时间的增长TLR4蛋白表达量递增；免疫荧光检测NF-κB的核转位情况，结果显示：与空白对照组相比，长春瑞滨干预1h后以及洗去长春瑞滨继续培养6h、12h，NF-κB的核转位率明显升高（P＜0.05）。

结论 1．小鼠尾部血管推注长春瑞滨可能造成静脉炎，出现程度不同的血管内皮细胞脱落、炎性细胞浸润、血栓形成等病理学改变。 2．小鼠以及HUVEC细胞试验表明在使用长春瑞滨导致药物性静脉炎过程中，内皮细胞TLR4表达增强；3．HUVEC细胞试验表明，VIN刺激上调HUVEC TLR4蛋白及其mRNA的表达。4．HUVEC细胞试验表明，VIN刺激促进HUVEC NF-κB的活化及核转位。5．上述研究表明，TLR4可能在长春瑞滨导致静脉炎发生中发挥着重要作用。

关键词 Toll样受体；长春瑞滨；静脉炎；人脐静脉内皮细胞；机制

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徐州医学院硕士论文

Effect Mechanism of TLR4 in vascular endothelial

injury that induced by Vinorelbine

Abstract

Objective To investigate the effect mechanism of TLR4 in vascular endothelial injury that induced by Vinorelbine.

Methods 1. 20 BALB/c mice were divided into to two groups, vinorelbine was injected in the experimental group. The same dose of saline was injected in the vehicle control group. Mice were euthanized 48 hours after vinorelbine injection, and injected mouse trail tissue near the puncturation site was fixed and further evaluated by pathology. Severity of phlebitis was scored and the expression of TLR4 in phlebitis tissues was measured by immunohistochemistry. 2. The cells, at a density of 5×104 cells/mL , were seeded in 25-cm2 flasks or 24-well plates. Upon reaching cell confluence of 90%-95%，the cells were washed twice with phosphate-buffered saline (PBS), and re-fed with VIN solution at the final concentration of 0.05 mg/L in the flasks or wells, Untreated control cells were re-fed with only medium. After 60 min incubation with VIN, the cells were washed twice with PBS and re-fed with medium without VIN and grown for 0, 6 or 12h. After 0, 6 or 12h, cells were washed twice with PBS, and harvested for PCR, Western blot and immunofluorescence analysis. Experiments were repeated at least three times.

Results 1. After vinorelbine injection, the vein endothenia cells were disrupted, thrombus and inflamatory cells were founded, and TLR4 was positive in endothenia cells. In control group mice, the vein endothenia cells were intact and TLR4 expression in endothenia cells was negative or very low. 2. VIN treated cells stretch, extend, irregular, ill-defined disorder, part of the cell suspensionnoff. Wash away the VIN to continue to foster 6h, 12h. The cell morphology gradually returned to normal;

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