通心络抑制TNF-α诱导的小鼠巨噬细胞增殖与迁移

发布时间：2018-08-06 15:13

【摘要】：目的：肿瘤坏死因子α(Tumor necrosis factor α，TNF-α)是一种具备多种生物学功能的强效细胞因子，主要由单核细胞、巨噬细胞、T细胞、自然杀伤(NK)细胞等免疫细胞产生。作为免疫和炎症反应的重要介质，TNF-α可以诱生其它细胞因子,诱导细胞的抗病毒活性,刺激血管形成以及成纤维细胞有丝分裂等。在生理状态下,它具有抗肿瘤、抗感染和促进组织修复等重要作用,对机体有利;一旦持续释放,则会引发炎症级联反应，造成机体发热、休克、恶病质和组织损伤。因此，抑制TNF-α介导的炎症反应，是治疗多种疾病的一个重要环节。通心络（TXL）含有水蛭、全蝎、蜈蚣、蝉蜕、土鳖虫等虫类药物和人参、赤芍等植物药物成分。研究证明,通心络具有改善血管内皮功能、舒张血管、溶栓、抗凝、调节血脂、稳定斑块、抗炎、抗氧化等功能。临床研究表明，通心络可以降低心绞痛患者体内C反应蛋白水平，同时能够抑制炎性因子产生。研究发现，通心络能够抑制IL-1β介导的冠状动脉血管壁的炎性反应，缓解内膜增生及减轻血管狭窄,其作用可能是通过下调炎性因子及黏附因子表达来实现的。巨噬细胞增殖及向血管壁迁移在血管炎性反应和内膜增生中发挥重要作用，然而，目前对TXL是否影响巨噬细胞的增殖和迁移尚不十分清楚，本研究探讨TNF-α对巨噬细胞增殖和迁移的影响，观察TXL是否可以抑制TNF-α诱导的小鼠巨噬细胞的炎症反应。方法：Real-time PCR和Western blot分析检测TNF-α对小鼠巨噬细胞RAW264.7cyclin D1、cyclin E1和mmp-2等基因表达的影响；伤口愈合实验检测RAW264.7细胞迁移能力。动物实验：实验动物分为对照和给药组，两组动物分别用水和TXL灌胃7天后，尾静脉注射TNF-α诱导炎症反应，取血检测血常规和C反应蛋白；取小鼠骨髓巨噬细胞检测cyclinD1、cyclin E1和mmp-2基因表达。结果： 1TNF-α诱导RAW264.7细胞cyclin D1和cyclin E1表达 Western blot分析结果显示，分别用0、1、10、100ng/ml TNF-α刺激RAW264.7细胞24h后，与对照组相比，cyclin D1和cyclin E1蛋白表达明显升高。用10ng/ml TNF-α分别刺激RAW264.7细胞0、6、12、24h后，cyclinD1和cyclin E1蛋白表达随刺激时间延长而逐渐上调。以上结果表明，TNF-α可剂量和时间依赖性的诱导增殖相关基因表达。 Real-time PCR结果也显示，与对照组相比，TNF-α刺激使cyclin D1和cyclin E1mRNA水平呈时间和剂量依赖性增加。这些结果表明, TNF-α通过诱导cyclin D1和cyclin E1表达促进RAW264.7细胞增殖。 2通心络抑制TNF-α诱导的RAW264.7细胞增殖用通心络（TXL）预孵育RAW264.7细胞24h后，再给予TNF-α刺激24h，检测cyclin D1和cyclin E1蛋白水平。Western blot结果显示，TXL预处理组较单纯TNF-α刺激组，cyclin D1和cyclin E1蛋白表达下调。Real-time PCR分析结果与Western blot结果一致。上述结果表明，TXL抑制TNF-α诱导的cyclin D1和cyclin E1表达。 3TNF-α诱导RAW264.7细胞mmp-2表达用不同浓度的TNF-α（0、1、10、100ng/ml）分别刺激RAW264.7细胞24h后，进行Western blot分析。结果显示，，随着TNF-α浓度增加，迁移相关基因mmp-2的表达水平呈剂量依赖性升高。用10ng/ml TNF-α刺激RAW264.7细胞0、6、12、24h后，随刺激时间延长，mmp-2表达呈时间依赖性增加。Real-time PCR分析结果与Western blot分析结果一致。上述结果表明，TNF-α通过诱导mmp-2表达促进RAW264.7细胞迁移。 4通心络抑制TNF-α诱导的RAW264.7细胞迁移用TXL预孵育RAW264.7细胞24h后，再给予TNF-α刺激24h，观察mmp-2表达水平。Western blot结果显示，TXL预处理组较单纯TNF-α刺激组，mmp-2表达水平下调。Real-time PCR分析结果与Western blot结果一致。伤口愈合实验结果显示，用TXL预孵育RAW264.7细胞24h后，再用10ng/ml TNF-α刺激24h，检查细胞迁移情况。HE染色结果显示，TXL可以抑制TNF-α诱导的RAW264.7细胞迁移。上述结果显示，TXL通过抑制mmp-2表达而抑制TNF-α诱导的RAW264.7细胞迁移。 5通心络抑制TNF-α诱导的炎症反应小鼠分为对照和给药两组，分别用水和TXL灌胃7天后，尾静脉注射TNF-α0.1mg/kg。检查结果显示，对照组小鼠体温、C反应蛋白、白细胞、中性粒细胞水平均明显升高。与对照组相比，给药组小鼠体温、C反应蛋白、白细胞、中性粒细胞均显著下降，骨髓巨噬细胞中cyclin D1、cyclin E1和mmp-2mRNA水平降低。以上结果表明，TXL通过抑制cyclinD1、cyclin E1和mmp-2表达，抑制TNF-α诱导的小鼠机体内炎性反应。结论： 1TNF-α促进RAW264.7细胞增殖相关基因cyclin D1和cyclin E1表达。 2通心络通过下调cyclin D1和cyclin E1表达，抑制TNF-α诱导的RAW264.7细胞增殖。 3TNF-α诱导RAW264.7细胞迁移相关基因mmp-2表达。 4通心络通过下调mmp-2表达，抑制TNF-α诱导的RAW264.7细胞迁移。
[Abstract]:Objective: Tumor necrosis factor alpha (TNF- alpha) is a powerful cytokine with many biological functions. It is mainly produced by immune cells, such as monocyte, macrophage, T cell, and natural killer (NK) cell. As an important medium for immune and inflammatory reaction, TNF- alpha can induce other cytokines and induce cells. Antiviral activity, stimulation of blood vessels and fibroblasts mitosis. In physiological state, it plays an important role in anti-tumor, anti infection and tissue repair, and is beneficial to the body. Once sustained release, it may cause inflammation cascade, causing body heat, shock, cachexia and tissue damage. Therefore, inhibition of TNF- a mediates. The inflammatory response is an important link in the treatment of many diseases.
Tongxinluo (TXL) contains leech, scorpion, centipede, cicada, woodlouse worm and other insect drugs and ginseng, Radix Paeoniae, and other plant drugs. The study shows that Tongxinluo has the functions of improving vascular endothelial function, diastolic blood vessel, thrombolytic, anticoagulant, regulating blood lipid, stabilizing plaque, anti-inflammatory, antioxidation and so on. Clinical study shows Tongxinluo can reduce angina patients The level of C reactive protein in the body can inhibit the production of inflammatory factors. It is found that Tongxinluo can inhibit the inflammatory response of the coronary artery wall mediated by IL-1 beta, alleviate intimal hyperplasia and reduce vascular stenosis. The effect may be achieved by down regulating the expression of inflammatory factors and adhesion factors. The proliferation of macrophages and the wall of blood vessels Migration plays an important role in vasculitis and intimal hyperplasia. However, it is not very clear whether TXL affects the proliferation and migration of macrophages. This study explores the effect of TNF- alpha on the proliferation and migration of macrophages and whether TXL can inhibit the inflammatory response of macrophages induced by TNF- alpha.
Methods: Real-time PCR and Western blot analysis were used to detect the effect of TNF- alpha on the gene expression of RAW264.7cyclin D1, cyclin E1 and MMP-2 in mouse macrophages. The wound healing test was used to detect the migration ability of RAW264.7 cells. Animal experiments were divided into control and administration groups. The two groups of animals were injected with water and TXL for 7 days, and the tail vein was injected. Inflammation was induced by TNF-alpha irradiation, and blood samples were taken to detect blood routine and C-reactive protein, and the expression of cyclin D1, cyclin E1 and MMP-2 genes in bone marrow macrophages of mice were detected.
Result:
1TNF- alpha induces the expression of cyclin D1 and cyclin E1 in RAW264.7 cells
The results of Western blot analysis showed that the expression of cyclin D1 and cyclin E1 increased significantly compared with the control group after 0,1,10100ng/ml TNF- alpha was used to stimulate 24h in RAW264.7 cells respectively.
After 10ng/ml TNF- alpha was used to stimulate 0,6,12,24h in RAW264.7 cells, the expression of cyclinD1 and cyclin E1 protein was gradually up-regulated with the time of stimulation. The above results showed that TNF- alpha could induce proliferation related gene expression in dose and time dependent manner.
Real-time PCR results also showed that TNF- alpha stimulation increased the time and dose dependence of cyclin D1 and cyclin E1mRNA levels compared with the control group. These results suggest that TNF- alpha promotes the proliferation of cells by inducing cyclin D1 and cyclin E1.
2 Tongxinluo inhibits proliferation of RAW264.7 cells induced by TNF- alpha
RAW264.7 cells 24h were incubated with Tongxinluo (TXL), and then 24h was stimulated by TNF- alpha, and cyclin D1 and cyclin E1 protein levels were detected at.Western blot. NF- - alpha induced expression of cyclin D1 and cyclin E1.
3TNF- alpha induces the expression of MMP-2 in RAW264.7 cells
Western blot analysis was performed after RAW264.7 cell 24h was stimulated with different concentrations of TNF- alpha (0,1,10100ng/ml). The results showed that the expression level of migration related gene MMP-2 increased in a dose-dependent manner with the increase of TNF- alpha concentration. The expression was time dependent with 10ng/ml TNF- alpha stimulation of RAW264.7 cell 0,6,12,24h. The results of Real-time PCR and Western blot showed that TNF-a promoted the migration of RAW264.7 cells by inducing MMP-2 expression.
4 Tongxinluo inhibits TNF- alpha induced RAW264.7 cell migration
After incubating RAW264.7 cell 24h with TXL, TNF- alpha was then given to 24h, and the results of MMP-2 expression level.Western blot showed that the TXL preconditioning group was more than the pure TNF- alpha stimulation group, and the MMP-2 expression level was down.
The results of the wound healing experiment showed that after incubating the RAW264.7 cell 24h with TXL, 10ng/ml TNF- alpha was used to stimulate 24h, and the cell migration of.HE staining results showed that TXL could inhibit the migration of RAW264.7 cells induced by TNF- alpha.
These results suggest that TXL inhibits TNF- alpha induced RAW264.7 cell migration by inhibiting MMP-2 expression.
5 Tongxinluo inhibits the inflammatory response induced by TNF- alpha
The mice were divided into two groups. After 7 days of water use and TXL gastric perfusion, the results of TNF- alpha 0.1mg/kg. in the tail vein showed that the body temperature, C reactive protein, leucocyte and neutrophils level in the control group were significantly increased. Compared with the control group, the body temperature, C reactive protein, leukocyte and neutrophils decreased significantly in the administration group. The levels of cyclin D1, cyclin E1 and mmp-2mRNA in medullary macrophages were reduced. The above results showed that TXL inhibited the inflammatory response in mice induced by TNF- alpha by inhibiting cyclinD1, cyclin E1 and MMP-2 expression.
Conclusion:
1TNF- alpha promotes RAW264.7 cell proliferation related genes cyclin D1 and cyclin E1 expression.
2 Tongxinluo inhibited the proliferation of RAW264.7 cells induced by TNF- alpha by down regulating the expression of cyclin D1 and cyclin E1.
3TNF- alpha induced RAW264.7 cell migration related gene MMP-2 expression.
4 Tongxinluo inhibited the migration of RAW264.7 cells induced by TNF- alpha by down regulating the expression of MMP-2.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R363

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