發(fā)布時間：2018-08-06 15:13

【摘要】：目的：腫瘤壞死因子α(Tumor necrosis factor α，TNF-α)是一種具備多種生物學(xué)功能的強效細(xì)胞因子，主要由單核細(xì)胞、巨噬細(xì)胞、T細(xì)胞、自然殺傷(NK)細(xì)胞等免疫細(xì)胞產(chǎn)生。作為免疫和炎癥反應(yīng)的重要介質(zhì)，TNF-α可以誘生其它細(xì)胞因子,誘導(dǎo)細(xì)胞的抗病毒活性,刺激血管形成以及成纖維細(xì)胞有絲分裂等。在生理狀態(tài)下,它具有抗腫瘤、抗感染和促進(jìn)組織修復(fù)等重要作用,對機體有利;一旦持續(xù)釋放,則會引發(fā)炎癥級聯(lián)反應(yīng)，造成機體發(fā)熱、休克、惡病質(zhì)和組織損傷。因此，抑制TNF-α介導(dǎo)的炎癥反應(yīng)，是治療多種疾病的一個重要環(huán)節(jié)。 通心絡(luò)（TXL）含有水蛭、全蝎、蜈蚣、蟬蛻、土鱉蟲等蟲類藥物和人參、赤芍等植物藥物成分。研究證明,通心絡(luò)具有改善血管內(nèi)皮功能、舒張血管、溶栓、抗凝、調(diào)節(jié)血脂、穩(wěn)定斑塊、抗炎、抗氧化等功能。臨床研究表明，通心絡(luò)可以降低心絞痛患者體內(nèi)C反應(yīng)蛋白水平，同時能夠抑制炎性因子產(chǎn)生。研究發(fā)現(xiàn)，通心絡(luò)能夠抑制IL-1β介導(dǎo)的冠狀動脈血管壁的炎性反應(yīng)，緩解內(nèi)膜增生及減輕血管狹窄,其作用可能是通過下調(diào)炎性因子及黏附因子表達(dá)來實現(xiàn)的。巨噬細(xì)胞增殖及向血管壁遷移在血管炎性反應(yīng)和內(nèi)膜增生中發(fā)揮重要作用，然而，目前對TXL是否影響巨噬細(xì)胞的增殖和遷移尚不十分清楚，本研究探討TNF-α對巨噬細(xì)胞增殖和遷移的影響，觀察TXL是否可以抑制TNF-α誘導(dǎo)的小鼠巨噬細(xì)胞的炎癥反應(yīng)。 方法：Real-time PCR和Western blot分析檢測TNF-α對小鼠巨噬細(xì)胞RAW264.7cyclin D1、cyclin E1和mmp-2等基因表達(dá)的影響；傷口愈合實驗檢測RAW264.7細(xì)胞遷移能力。動物實驗：實驗動物分為對照和給藥組，兩組動物分別用水和TXL灌胃7天后，尾靜脈注射TNF-α誘導(dǎo)炎癥反應(yīng)，取血檢測血常規(guī)和C反應(yīng)蛋白；取小鼠骨髓巨噬細(xì)胞檢測cyclinD1、cyclin E1和mmp-2基因表達(dá)。 結(jié)果： 1TNF-α誘導(dǎo)RAW264.7細(xì)胞cyclin D1和cyclin E1表達(dá) Western blot分析結(jié)果顯示，分別用0、1、10、100ng/ml TNF-α刺激RAW264.7細(xì)胞24h后，與對照組相比，cyclin D1和cyclin E1蛋白表達(dá)明顯升高。 用10ng/ml TNF-α分別刺激RAW264.7細(xì)胞0、6、12、24h后，cyclinD1和cyclin E1蛋白表達(dá)隨刺激時間延長而逐漸上調(diào)。以上結(jié)果表明，TNF-α可劑量和時間依賴性的誘導(dǎo)增殖相關(guān)基因表達(dá)。 Real-time PCR結(jié)果也顯示，與對照組相比，TNF-α刺激使cyclin D1和cyclin E1mRNA水平呈時間和劑量依賴性增加。這些結(jié)果表明, TNF-α通過誘導(dǎo)cyclin D1和cyclin E1表達(dá)促進(jìn)RAW264.7細(xì)胞增殖。 2通心絡(luò)抑制TNF-α誘導(dǎo)的RAW264.7細(xì)胞增殖 用通心絡(luò)（TXL）預(yù)孵育RAW264.7細(xì)胞24h后，再給予TNF-α刺激24h，檢測cyclin D1和cyclin E1蛋白水平。Western blot結(jié)果顯示，TXL預(yù)處理組較單純TNF-α刺激組，cyclin D1和cyclin E1蛋白表達(dá)下調(diào)。Real-time PCR分析結(jié)果與Western blot結(jié)果一致。上述結(jié)果表明，TXL抑制TNF-α誘導(dǎo)的cyclin D1和cyclin E1表達(dá)。 3TNF-α誘導(dǎo)RAW264.7細(xì)胞mmp-2表達(dá) 用不同濃度的TNF-α（0、1、10、100ng/ml）分別刺激RAW264.7細(xì)胞24h后，進(jìn)行Western blot分析。結(jié)果顯示，，隨著TNF-α濃度增加，遷移相關(guān)基因mmp-2的表達(dá)水平呈劑量依賴性升高。用10ng/ml TNF-α刺激RAW264.7細(xì)胞0、6、12、24h后，隨刺激時間延長，mmp-2表達(dá)呈時間依賴性增加。Real-time PCR分析結(jié)果與Western blot分析結(jié)果一致。上述結(jié)果表明，TNF-α通過誘導(dǎo)mmp-2表達(dá)促進(jìn)RAW264.7細(xì)胞遷移。 4通心絡(luò)抑制TNF-α誘導(dǎo)的RAW264.7細(xì)胞遷移 用TXL預(yù)孵育RAW264.7細(xì)胞24h后，再給予TNF-α刺激24h，觀察mmp-2表達(dá)水平。Western blot結(jié)果顯示，TXL預(yù)處理組較單純TNF-α刺激組，mmp-2表達(dá)水平下調(diào)。Real-time PCR分析結(jié)果與Western blot結(jié)果一致。 傷口愈合實驗結(jié)果顯示，用TXL預(yù)孵育RAW264.7細(xì)胞24h后，再用10ng/ml TNF-α刺激24h，檢查細(xì)胞遷移情況。HE染色結(jié)果顯示，TXL可以抑制TNF-α誘導(dǎo)的RAW264.7細(xì)胞遷移。 上述結(jié)果顯示，TXL通過抑制mmp-2表達(dá)而抑制TNF-α誘導(dǎo)的RAW264.7細(xì)胞遷移。 5通心絡(luò)抑制TNF-α誘導(dǎo)的炎癥反應(yīng) 小鼠分為對照和給藥兩組，分別用水和TXL灌胃7天后，尾靜脈注射TNF-α0.1mg/kg。檢查結(jié)果顯示，對照組小鼠體溫、C反應(yīng)蛋白、白細(xì)胞、中性粒細(xì)胞水平均明顯升高。與對照組相比，給藥組小鼠體溫、C反應(yīng)蛋白、白細(xì)胞、中性粒細(xì)胞均顯著下降，骨髓巨噬細(xì)胞中cyclin D1、cyclin E1和mmp-2mRNA水平降低。以上結(jié)果表明，TXL通過抑制cyclinD1、cyclin E1和mmp-2表達(dá)，抑制TNF-α誘導(dǎo)的小鼠機體內(nèi)炎性反應(yīng)。 結(jié)論： 1TNF-α促進(jìn)RAW264.7細(xì)胞增殖相關(guān)基因cyclin D1和cyclin E1表達(dá)。 2通心絡(luò)通過下調(diào)cyclin D1和cyclin E1表達(dá)，抑制TNF-α誘導(dǎo)的RAW264.7細(xì)胞增殖。 3TNF-α誘導(dǎo)RAW264.7細(xì)胞遷移相關(guān)基因mmp-2表達(dá)。 4通心絡(luò)通過下調(diào)mmp-2表達(dá)，抑制TNF-α誘導(dǎo)的RAW264.7細(xì)胞遷移。
[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.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R363

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