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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徐州医学院硕士论文
To verify TLR4 mRNA expression changes in the HUVEC, RT-PCR and quantitative real time PCR were performed. The TLR4 mRNA expression levels were significantly increased in the VIN-treated HUVEC (P<0.05), compared with vehicle-treated group. The effect of VIN reached a maximum at the time after exposure 1h; We performed western blot for the relative protein expression of TLR4 in HUVECs to assess the effect after treatment with VIN. The expression of TLR4 protein in all VIN-treated groups was increased significantly compared with vehicle-treated group(P<0.05); NF-κB activation as determined by NF-κB phosphorylation was detected at different time and was no longer detectable at later time points. As expected, nuclear translocation of p65 occurred in HUVEC treated with VIN, and NF-κB activation occurred within a very narrow window in HUVEC during the course of vascular endothelial injury.
Conclusions 1. Pathohistological changes of the model group were observed, such as loss of venous endothelial cells, inflammatory cell infiltration and thrombus. 2. mouse and cell experiments indicates that TLR4 expression is up-regulated in vascular endothelial injury that induced by Vinorelbine. 3. cell experiment indicates that TLR4 mRNA and protein are up-regulated by Vinorelbine in HUVEC. 4. cell experiment indicates that nuclear translocation of p65 occurred in HUVEC treated with VIN. 5. Tegether, our results suggest that TLR4 may play an important role in the development of vascular endothelial injury that induced by Vinorelbine.
Key words Toll-like receptor; Vinorelbine; Phlebitis; HUVEC; Mechanism
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徐州医学院硕士论文
前 言
随着工业化的进展、环境污染的加剧，人们的生活节奏越来越快，不良生活习惯及不良饮食的影响，肿瘤的发病率也逐年增高，无论在发达国家还是发展中国家，恶性肿瘤的危害都不容忽视。静脉输注抗肿瘤药物是治疗恶性肿瘤的主要方法之一，但往往引起严重的毒副反应，除对造血系统及全身重要脏器具有毒性以外，对血管周围组织还可引起严重的炎性刺激反应。局部组织表现红、肿、热、痛等炎症反应，有时沿静脉走向出现条索状改变，严重者局部组织糜烂、坏死。
肿瘤患者长期放、化疗，机体免疫力低下时，常导致全身感染而危及生命。
长春瑞滨是临床常见的抗肿瘤药物，属于长春花生物碱类药物，它的抗肿瘤活性主要通过干扰微管蛋白而抑制中期有丝分裂，目前主要用于非小细胞肺癌以及其他实体瘤等。然而在长春瑞滨给病人带来抗肿瘤疗效的同时，它作为中度发疱剂的一种，也常常引起局部毒副反应，局部组织发红，疼痛、肿胀甚至糜烂坏死。据报道[1]，其致静脉炎的发生率可达24-33%，国内学者报道其发生率甚至可达87%[2]。虽然这种药物性静脉炎在临床常见，但是其确切机制仍不明确，目前普遍的观点认为药物性静脉炎是由于药物对血管内皮细胞的化学性刺激而引起的无菌性炎症。
药物性静脉炎的形成因素包括药物种类、药液的温度、药液的PH值、药液的渗透压、药物本身的毒性作用、药物引起的变态反应以及药液的输液微粒等。另外药物性的静脉炎发生还与输入药液的剂量、浓度、输入速度、时间以及输注方法等有关[2-28]。抗生素和抗肿瘤药物导致的静脉炎的发生率最高[29-30]，抗肿瘤药物中以长春瑞滨最为严重。尽管临床工作者采取了许多防范措施，例如给药部位热敷、冷敷的方法，以及局部给予糖皮质激素、非甾体抗炎药、抗组胺药、血管扩张剂、局部麻醉药、抗凝药、溶栓药甚至活血化瘀类中药等，但遗憾的是由
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