本文選題：腫瘤壞死因子樣凋亡微弱誘導(dǎo)劑 + 心肌成纖維細胞��； 參考：《山東大學(xué)》2012年碩士論文

【摘要】：背景： 心肌纖維化(myocardial fibrosis,MF)主要表現(xiàn)為心肌成纖維細胞(cardiac fibroblasts, CFs)數(shù)目的增多和心肌細胞外間質(zhì)膠原的過度沉積,可存在于多種心血管系統(tǒng)疾病。心肌纖維化造成心肌僵硬度增加,使心室的收縮和舒張功能下降,并影響心肌電生理,可導(dǎo)致心力衰竭、心律失常和心源性猝死等并發(fā)癥,嚴重危害人類身體健康。心肌纖維化是高血壓性心臟病的主要病理基礎(chǔ)之一。近年來,隨著高血壓病發(fā)病率的增高,高血壓性心肌纖維化的發(fā)生機制和預(yù)防成為國內(nèi)外研究的熱點。 高血壓心肌纖維化的病理生理過程是非常復(fù)雜的。近年來研究發(fā)現(xiàn),炎癥反應(yīng)在其病理過程中發(fā)揮了重要的作用,MF被認為是高血壓、激素異常分泌和炎癥反應(yīng)之間反復(fù)相互作用的結(jié)果,是一個慢性炎癥反應(yīng)過程。 腫瘤壞死因子樣凋亡微弱誘導(dǎo)劑(tumor necrosis factor-like weak inducer of apoptosis, TWEAK)是腫瘤壞死因子(tumor necrosis factor, TNF)超家族的新成員,可在多種組織細胞中表達。TWEAK除具備TNF超家族的共同結(jié)構(gòu)特征和對腫瘤細胞有殺傷作用外,還有調(diào)節(jié)細胞的生長、增殖及凋亡,促炎性反應(yīng)及血管生成等作用。TWEAK能夠通過刺激細胞分泌多種炎性因子,促進心肌成纖維細胞的增殖和膠原表達增多,從而促進心肌纖維化的發(fā)生和發(fā)展,但其機制尚不明確。研究發(fā)現(xiàn),核轉(zhuǎn)錄因子NF-κB是介導(dǎo)血管緊張素II (angiotensin II, Ang II)、 TNF-a促心肌成纖維細胞增殖和膠原表達作用的關(guān)鍵因子�；|(zhì)金屬蛋白酶9(matrixmetallopeptidase9, MMP9)被證實在細胞外基質(zhì)重塑及心肌纖維化過程中起主要作用。但TWEAK與NF-κB, MMP9在心肌纖維化中的關(guān)系尚未見報道。 目的： 研究腫瘤壞死因子樣凋亡微弱誘導(dǎo)劑(TWEAK)對大鼠心肌成纖維細胞NF-κB通路的影響及作用機制,探討NF-κB、 MMP9等因子與心肌纖維化的關(guān)系。 方法： 1.采用胰酶消化法培養(yǎng)新生Wistar大鼠心肌成纖維細胞,倒置顯微鏡觀察大鼠心肌成纖維細胞的形態(tài),波形蛋白免疫熒光法鑒定心肌成纖維細胞。實驗選用第2-4代細胞。 2.采用qRT-PCR法檢測兩組NF-κB和MMP9基因表達 實驗共分兩組,對照組：不加干預(yù)因素；TWEAK組：加入rhTWEAK,使其濃度達100μg/L。繼續(xù)培養(yǎng)6h后分別提取RNA檢測NF-κB的表達情況,24h后分別提取RNA檢測MMP9的表達情況。 3.采用Western blot法檢測NF-κB和MMP9蛋白表達 TWEAK干預(yù)后檢測NF-κB的蛋白表達：①濃度梯度組：按rhTWEAK的終濃度分為對照組(0μg/L)、1μg/L組、10μg/L組、100μg/L組和200μg/L組,繼續(xù)孵育8h后提取核蛋白；②時間梯度組：培養(yǎng)液換為含100μg/L rhTWEAK的無血清DMEM繼續(xù)孵育,按分組不同分別孵育0、3、6、9、12h后提取核蛋白。 TWEAK和PDTC干預(yù)后檢測NF-κB蛋白的表達：①TWEAK組：培養(yǎng)液換為含100μg/L rhTWEAK的無血清DMEM,繼續(xù)孵育6h后提取核蛋白；②TWEAK+PDTC組：培養(yǎng)液換為含100μg/L rhTWEAK和100μmol/L PDTC的無血清DMEM,繼續(xù)孵育6h后提取核蛋白。 TWEAK和PDTC干預(yù)后檢測MMP9的蛋白表達：TWEAK+PDTC組：培養(yǎng)液換為含100μg/L rhTWEAK和100μmol/L PDTC的無血清DMEM,繼續(xù)孵育24h后提取蛋白；②刺激組：換含100μg/L rhTWEAK的無血清DMEM,繼續(xù)孵育24h后提取蛋白；③對照組：只加無血清DMEM,繼續(xù)孵育24h后提取蛋白。 4.四甲基偶氮唑藍(MTT)法測定TWEAK和PDTC干預(yù)后細胞增殖情況 培養(yǎng)CFs至指數(shù)生長期,調(diào)整細胞密度為5×103個/孔接種于96孔培養(yǎng)板中,培養(yǎng)24h后,換DMEM培養(yǎng)液繼續(xù)培養(yǎng),24h后分組：①TWEAK+PDTC組：換含100ug/L rhTWEAK和100umol/L PDTC的DMEM;②TWEAK組：換含100ug/LrhTWEAK的DMEM;③對照組：只加DMEM.繼續(xù)培養(yǎng)48h后,MTT法測定各組細胞增殖情況。 結(jié)果： 1.qRT-PCR檢測NF-κB和MMP9的表達,刺激組NF-κB(5.3517)和MMP9(5.8971) mRNA表達水平顯著高于對照組(P0.01)。 2.不同濃度的rhTWEAK刺激8h后,NF-κB蛋白表達呈現(xiàn)劑量依賴性增加。對照組(0.510±0.011)、1μg/L組(0.860±0.069)、10μg/L組(1.224±0.010)、100μg/L組(1.908±0.055)表達依次增加(n=3)(P0.01)。而200μg/L rhTWEAK組(1.942±0.069)與100μg/L組相比未見顯著性變化(P0.05)。 3.100μg/L rhTWEAK刺激3h后NF-κB蛋白表達(0.815±0.063)開始增加,6h后(1.426±0.056)達到最大值,12h后(0.456±0.048)明顯衰減(n=3)(P0.01)。 4.100ug/L rhTWEAK作用24h后,與對照組(0.232±0.010)相比,刺激組(0.871±0.018)MMP9表達明顯增加；用100umol/L PDTC抑制NF-κB表達后,TWEAK+PDTC組(0.422±0.022)MMP9表達較刺激組顯著減少(n=3)(P0.01)。 5.100ug/L rhTWEAK作用后刺激組A492值顯著高于對照組(P0.01),用100umol/LPDTC抑制NF-κB后,TWEAK+PDTC組A492值明顯降低(P0.05)。 結(jié)論： 1. rhTWEAK可促進大鼠成纖維細胞的NF-κB和MMP9mRNA表達上調(diào),蛋白表達明顯增加,同時促進心肌成纖維細胞增殖。NF-κB蛋白表達對rhTWEAK的刺激呈劑量依賴性關(guān)系,濃度為100μg/L時達到最大值；rhTWEAK刺激后6h后,NF-κB蛋白表達達到最大值,12h后明顯衰減,rhTWEAK呈作用時間依賴性關(guān)系。 2. NF-κB抑制劑PDTC可明顯減弱由TWEAK介導(dǎo)的MMP9蛋白表達和細胞增殖效應(yīng),表明TWEAK促進心肌纖維化的作用依賴于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.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R329.2

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