TWEAK通过NF-κB途径促进大鼠心肌成纤维细胞增殖并表达基质金属蛋白酶9

发布时间：2018-06-30 16:50

本文选题：肿瘤坏死因子样凋亡微弱诱导剂 + 心肌成纤维细胞　；参考：《山东大学》2012年硕士论文

【摘要】：背景：心肌纤维化(myocardial fibrosis,MF)主要表现为心肌成纤维细胞(cardiac fibroblasts, CFs)数目的增多和心肌细胞外间质胶原的过度沉积,可存在于多种心血管系统疾病。心肌纤维化造成心肌僵硬度增加,使心室的收缩和舒张功能下降,并影响心肌电生理,可导致心力衰竭、心律失常和心源性猝死等并发症,严重危害人类身体健康。心肌纤维化是高血压性心脏病的主要病理基础之一。近年来,随着高血压病发病率的增高,高血压性心肌纤维化的发生机制和预防成为国内外研究的热点。高血压心肌纤维化的病理生理过程是非常复杂的。近年来研究发现,炎症反应在其病理过程中发挥了重要的作用,MF被认为是高血压、激素异常分泌和炎症反应之间反复相互作用的结果,是一个慢性炎症反应过程。肿瘤坏死因子样凋亡微弱诱导剂(tumor necrosis factor-like weak inducer of apoptosis, TWEAK)是肿瘤坏死因子(tumor necrosis factor, TNF)超家族的新成员,可在多种组织细胞中表达。TWEAK除具备TNF超家族的共同结构特征和对肿瘤细胞有杀伤作用外,还有调节细胞的生长、增殖及凋亡,促炎性反应及血管生成等作用。TWEAK能够通过刺激细胞分泌多种炎性因子,促进心肌成纤维细胞的增殖和胶原表达增多,从而促进心肌纤维化的发生和发展,但其机制尚不明确。研究发现,核转录因子NF-κB是介导血管紧张素II (angiotensin II, Ang II)、 TNF-a促心肌成纤维细胞增殖和胶原表达作用的关键因子。基质金属蛋白酶9(matrixmetallopeptidase9, MMP9)被证实在细胞外基质重塑及心肌纤维化过程中起主要作用。但TWEAK与NF-κB, MMP9在心肌纤维化中的关系尚未见报道。目的：研究肿瘤坏死因子样凋亡微弱诱导剂(TWEAK)对大鼠心肌成纤维细胞NF-κB通路的影响及作用机制,探讨NF-κB、 MMP9等因子与心肌纤维化的关系。方法： 1.采用胰酶消化法培养新生Wistar大鼠心肌成纤维细胞,倒置显微镜观察大鼠心肌成纤维细胞的形态,波形蛋白免疫荧光法鉴定心肌成纤维细胞。实验选用第2-4代细胞。 2.采用qRT-PCR法检测两组NF-κB和MMP9基因表达实验共分两组,对照组：不加干预因素；TWEAK组：加入rhTWEAK,使其浓度达100μg/L。继续培养6h后分别提取RNA检测NF-κB的表达情况,24h后分别提取RNA检测MMP9的表达情况。 3.采用Western blot法检测NF-κB和MMP9蛋白表达 TWEAK干预后检测NF-κB的蛋白表达：①浓度梯度组：按rhTWEAK的终浓度分为对照组(0μg/L)、1μg/L组、10μg/L组、100μg/L组和200μg/L组,继续孵育8h后提取核蛋白；②时间梯度组：培养液换为含100μg/L rhTWEAK的无血清DMEM继续孵育,按分组不同分别孵育0、3、6、9、12h后提取核蛋白。 TWEAK和PDTC干预后检测NF-κB蛋白的表达：①TWEAK组：培养液换为含100μg/L rhTWEAK的无血清DMEM,继续孵育6h后提取核蛋白；②TWEAK+PDTC组：培养液换为含100μg/L rhTWEAK和100μmol/L PDTC的无血清DMEM,继续孵育6h后提取核蛋白。 TWEAK和PDTC干预后检测MMP9的蛋白表达：TWEAK+PDTC组：培养液换为含100μg/L rhTWEAK和100μmol/L PDTC的无血清DMEM,继续孵育24h后提取蛋白；②刺激组：换含100μg/L rhTWEAK的无血清DMEM,继续孵育24h后提取蛋白；③对照组：只加无血清DMEM,继续孵育24h后提取蛋白。 4.四甲基偶氮唑蓝(MTT)法测定TWEAK和PDTC干预后细胞增殖情况培养CFs至指数生长期,调整细胞密度为5×103个/孔接种于96孔培养板中,培养24h后,换DMEM培养液继续培养,24h后分组：①TWEAK+PDTC组：换含100ug/L rhTWEAK和100umol/L PDTC的DMEM;②TWEAK组：换含100ug/LrhTWEAK的DMEM;③对照组：只加DMEM.继续培养48h后,MTT法测定各组细胞增殖情况。结果： 1.qRT-PCR检测NF-κB和MMP9的表达,刺激组NF-κB(5.3517)和MMP9(5.8971) mRNA表达水平显著高于对照组(P0.01)。 2.不同浓度的rhTWEAK刺激8h后,NF-κB蛋白表达呈现剂量依赖性增加。对照组(0.510±0.011)、1μg/L组(0.860±0.069)、10μg/L组(1.224±0.010)、100μg/L组(1.908±0.055)表达依次增加(n=3)(P0.01)。而200μg/L rhTWEAK组(1.942±0.069)与100μg/L组相比未见显著性变化(P0.05)。 3.100μg/L rhTWEAK刺激3h后NF-κB蛋白表达(0.815±0.063)开始增加,6h后(1.426±0.056)达到最大值,12h后(0.456±0.048)明显衰减(n=3)(P0.01)。 4.100ug/L rhTWEAK作用24h后,与对照组(0.232±0.010)相比,刺激组(0.871±0.018)MMP9表达明显增加；用100umol/L PDTC抑制NF-κB表达后,TWEAK+PDTC组(0.422±0.022)MMP9表达较刺激组显著减少(n=3)(P0.01)。 5.100ug/L rhTWEAK作用后刺激组A492值显著高于对照组(P0.01),用100umol/LPDTC抑制NF-κB后,TWEAK+PDTC组A492值明显降低(P0.05)。结论： 1. rhTWEAK可促进大鼠成纤维细胞的NF-κB和MMP9mRNA表达上调,蛋白表达明显增加,同时促进心肌成纤维细胞增殖。NF-κB蛋白表达对rhTWEAK的刺激呈剂量依赖性关系,浓度为100μg/L时达到最大值；rhTWEAK刺激后6h后,NF-κB蛋白表达达到最大值,12h后明显衰减,rhTWEAK呈作用时间依赖性关系。 2. NF-κB抑制剂PDTC可明显减弱由TWEAK介导的MMP9蛋白表达和细胞增殖效应,表明TWEAK促进心肌纤维化的作用依赖于NF-κB通道的激活。
[Abstract]:Background:
Myocardial fibrosis (MF) is mainly manifested in the increase in the number of myocardial fibroblasts (cardiac fibroblasts, CFs) and the excessive deposition of collagen in the extracellular matrix of cardiac myocytes. It can exist in a variety of cardiovascular diseases. Myocardial fibrosis leads to the increase of myocardial stiffness, the decrease of ventricular systolic and diastolic function, and the influence of heart. Electromyography, which can lead to complications such as heart failure, arrhythmia and sudden cardiac death, seriously endangers human health. Myocardial fibrosis is one of the main pathological bases of hypertensive heart disease. In recent years, the pathogenesis and prevention of hypertensive myocardial fibrosis have become a study at home and abroad with the increase of the incidence of hypertension. Hotspot.
The pathophysiological process of hypertensive myocardial fibrosis is very complex. In recent years, it has been found that inflammation plays an important role in its pathological process. MF is considered to be the result of repeated interaction between hypertension, hormone abnormal secretion and inflammatory response, and is a slow inflammatory reaction process.
Tumor necrosis factor-like weak inducer of apoptosis, TWEAK) is a new member of the tumor necrosis factor (tumor necrosis factor, TNF) superfamily, which can be expressed in a variety of tissues and cells, except for the common structural features of the superfamily and the killing effect on the tumor cells. Regulation of cell growth, proliferation and apoptosis, proinflammatory response and angiogenesis,.TWEAK can stimulate cells to secrete a variety of inflammatory factors, promote the proliferation of myocardial fibroblasts and increase the expression of collagen, and thus promote the development and development of myocardial fibrosis, but the mechanism is not clear. The study found that the nuclear transcription factor NF- kappa B is Mediating the role of angiotensin II (angiotensin II, Ang II), TNF-a promoting the proliferation and collagen expression of myocardial fibroblasts. Matrix metalloproteinase 9 (matrixmetallopeptidase9, MMP9) has been proved to play a major role in the process of extracellular matrix remodeling and myocardial fibrosis. But TWEAK and NF- kappa B, MMP9 in myocardial fibrosis The relationship has not yet been reported.
Objective:
To study the effect of tumor necrosis factor like apoptosis weak inducer (TWEAK) on the NF- kappa B pathway of rat myocardial fibroblasts and its mechanism, and to explore the relationship between NF- kappa B, MMP9 and other factors and myocardial fibrosis.
Method:
1. the myocardial fibroblasts of neonatal Wistar rats were cultured by trypsin digestion method. The morphology of rat myocardial fibroblasts was observed by inverted microscope and the cardiac fibroblasts were identified by vimentin immunofluorescence. The 2-4 generation cells were selected in the experiment.
2. qRT-PCR method was used to detect NF- kappa B and MMP9 gene expression in two groups.
The experiment was divided into two groups, and the control group: no intervention factors; group TWEAK: adding rhTWEAK, the concentration reached 100 u g/L. and continued to be cultured for 6h, then the expression of NF- kappa B was detected by RNA, and the expression of RNA detection MMP9 was extracted respectively after 24h.
3. Western blot assay was used to detect NF- kappa B and MMP9 protein expression.
After TWEAK intervention, the protein expression of NF- kappa B was detected: (1) concentration gradient group: the concentration gradient group was divided into control group (0 mu g/L), 1 mu g/L group, 10 mu g/L group, 100 mu g/L group and 200 micron g/L group, and then incubated 8h after incubating 8h. After incubation of 0,3,6,9,12h, the nucleoprotein was extracted.
After the intervention of TWEAK and PDTC, the expression of NF- kappa B protein was detected: (1) TWEAK group: the culture medium was changed into serum-free DMEM containing 100 mu g/L rhTWEAK, and then incubated for 6h to extract nucleoprotein; (2) TWEAK+PDTC group: the culture solution was replaced by a serum containing 100 mu g/L rhTWEAK and 100 micron, and then incubated to extract the nucleoprotein.
The protein expression of MMP9 was detected after the intervention of TWEAK and PDTC: TWEAK+PDTC group: the culture medium was changed into 100 micron g/L rhTWEAK and 100 mol/L PDTC without serum DMEM, and then incubated for 24h after 24h. After 24h, the protein was extracted.
4. four methyl azolium blue (MTT) method was used to determine the proliferation of TWEAK and PDTC after intervention.
Culture CFs to exponential growth period, adjust cell density to 5 x 103 / hole inoculated in 96 hole culture plate, after culture 24h, change DMEM culture solution to continue culture, 24h group: 1 TWEAK+PDTC group: 100ug/L rhTWEAK and 100umol/L PDTC DMEM; second TWEAK group: substitute 100ug/LrhTWEAK TT assay was used to determine the cell proliferation.
Result:
1.qRT-PCR detected the expression of NF- kappa B and MMP9, and the expression level of NF- kappa B (5.3517) and MMP9 (5.8971) mRNA in stimulation group was significantly higher than that in control group (P0.01).
2. after 8h with different concentrations of rhTWEAK, the expression of NF- kappa B protein showed a dose-dependent increase. The control group (0.510 + 0.011), 1 mu g/L group (0.860 + 0.069), 10 mu g/L group (1.224 + 0.010), 100 mu g/L group (1.908 + 0.055) increased (n=3) (P0.01) in turn, while there was no significant change in 200 mu g/L rhTWEAK group compared with the g/L group.
After 3.100 mu g/L rhTWEAK stimulated 3h, the expression of NF- kappa B protein (0.815 + 0.063) began to increase. After 6h (1.426 + 0.056), the maximum value was reached, and (0.456 + 0.048) after 12h (n=3) (P0.01).
After the effect of 4.100ug/L rhTWEAK on 24h, the expression of (0.871 + 0.018) MMP9 in the stimulation group was significantly increased compared with the control group (0.232 + 0.010), and the expression of TWEAK+PDTC group (0.422 + 0.022) MMP9 was significantly lower than that of the stimulation group (n=3) (P0.01) after the expression of NF- kappa B with 100umol/L PDTC.
After 5.100ug/L rhTWEAK treatment, the A492 value of the stimulation group was significantly higher than that of the control group (P0.01), and the A492 value of TWEAK+PDTC group decreased significantly after 100umol/LPDTC inhibited NF- (B).
Conclusion:
1. rhTWEAK can increase the expression of NF- kappa B and MMP9mRNA in rat fibroblasts, increase the expression of protein, and promote the expression of.NF- kappa B protein expression to rhTWEAK in a dose-dependent manner, and reach the maximum value when the concentration is 100 mu g/L. After rhTWEAK, NF- kappa B protein expression reaches the maximum value after 6h. RhTWEAK markedly decreased after the treatment.
2. NF- kappa B inhibitor PDTC significantly weakened the expression of MMP9 protein and cell proliferation mediated by TWEAK, indicating that the role of TWEAK to promote myocardial fibrosis is dependent on the activation of the NF- kappa B channel.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329.2

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