本文選題：硫化氫 + 人臍靜脈內(nèi)皮細(xì)胞�。� 參考：《南華大學(xué)》2012年碩士論文

【摘要】：背景與目的： 隨著年齡的增加，機(jī)體各組織器官功能出現(xiàn)退行性改變，導(dǎo)致各種老年性疾病的發(fā)生。衰老已被認(rèn)為是心血管疾病發(fā)生的一個重要的危險(xiǎn)因素。內(nèi)皮是最容易受衰老影響的組織之一，內(nèi)皮細(xì)胞衰老是內(nèi)皮衰老的基礎(chǔ)。研究內(nèi)皮細(xì)胞衰老機(jī)制并探索減緩內(nèi)皮細(xì)胞衰老的新方法，對心血管疾病的防治具有重要意義。自由基學(xué)說認(rèn)為氧化應(yīng)激損傷是導(dǎo)致細(xì)胞衰老的主要原因之一。本研究采用過氧化氫（hydrogen peroxide，H_2O_2）誘導(dǎo)建立人臍靜脈內(nèi)皮細(xì)胞（human umbilicalvascular endothelial cells，HUVECs）衰老模型，旨在探討硫化氫（hydrogen sulfide，H2S）對HUVECs衰老的影響及其機(jī)制。 方法與結(jié)果： 1.采用酶消化法分離HUVECs，并進(jìn)行原代培養(yǎng)，選取第二代穩(wěn)定生長的細(xì)胞，倒置顯微鏡下觀察，細(xì)胞呈短梭形，邊界清晰，細(xì)胞經(jīng)體外培養(yǎng)7天后，細(xì)胞融合呈現(xiàn)典型的鋪路石樣排列。采用免疫組織化學(xué)法檢測HUVECs特異性標(biāo)記物Ⅷ因子的表達(dá)以鑒定HUVECs及其純度。 2.采用25μmol/L H_2O_2誘導(dǎo)HUVECs建立細(xì)胞衰老模型，應(yīng)用相差光鏡觀察細(xì)胞形態(tài)，流式細(xì)胞術(shù)檢測細(xì)胞周期，衰老相關(guān)β-半乳糖苷酶（senescenceassociated acidic-β-galactosidase， SA-β-gal）染色檢測衰老細(xì)胞率，蛋白免疫印跡（Western Blot）檢測細(xì)胞周期調(diào)控因子p21的表達(dá)，，從而鑒定HUVECs的衰老程度。結(jié)果表明，H_2O_2處理后，細(xì)胞體積增大，細(xì)胞邊界不清，大小不均，呈衰老細(xì)胞樣形態(tài)；SA-β-gal陽性細(xì)胞數(shù)達(dá)到11.2±1.06%（P0.05）；G0/G1期細(xì)胞比例增加25%；細(xì)胞周期蛋白p21的表達(dá)也明顯上調(diào)，為正常細(xì)胞的1.5倍，且差異具有統(tǒng)計(jì)學(xué)意義（P0.05）。 3.選擇不同濃度NaHS（0、15、30、60μmol/L）預(yù)先處理HUVECs48h后，再用H_2O_2誘導(dǎo)HUVECs衰老，采用SA-β-gal染色，流式細(xì)胞術(shù)檢測細(xì)胞周期變化，MTT法檢測細(xì)胞增殖率，劃痕實(shí)驗(yàn)檢測細(xì)胞遷移率，分析H2S對H_2O_2誘導(dǎo)的HUVECs衰老的影響。結(jié)果顯示，與H_2O_2單獨(dú)處理組比較，NaHS組SA-β-gal染色陽性細(xì)胞數(shù)隨濃度的增高而減少（P0.05），且NaHS濃度為60μmol/L時陽性細(xì)胞數(shù)最少(P0.01)；G0/G1期細(xì)胞比例降低16%，S期細(xì)胞比例增加14%（P0.05）；HUVECs的增殖遷移功能增加。 4.采用Western Blot及Sirt1活性檢測試劑盒測定H2S抗H_2O_2誘導(dǎo)HUVECs衰老中Sirt1蛋白表達(dá)及其活性，分析H2S對Sirt1的調(diào)節(jié)機(jī)制，結(jié)果表明，與H_2O_2相比，NaHS組Sirt1蛋白的表達(dá)沒有明顯改變，Sirt1的活性上調(diào)了19.21%。 5.采用沉默信息調(diào)節(jié)因子1（silent information regulator1， Sirt1）抑制劑NAM與NaHS共同孵育HUVECs48h，再應(yīng)用H_2O_2誘導(dǎo)HUVECs衰老，觀察HUVECs SA-β-gal染色陽性細(xì)胞數(shù)的改變以及HUVECs增殖、遷移能力的變化，分析Sirt1被抑制后， H2S抗H_2O_2誘導(dǎo)HUVECs衰老的作用是否發(fā)生變化。結(jié)果顯示，與NaHS組相比較，NAM+NaHS組SA-β-gal染色陽性細(xì)胞數(shù)增加了50%，并且HUVECs增殖，遷移功能明顯降低。 結(jié)論： H2S可拮抗H_2O_2誘導(dǎo)的HUVECs的衰老，其作用機(jī)制與上調(diào)Sirt1的活性有關(guān)。
[Abstract]:Background and purpose: With the increase of age, degenerative changes in the function of various tissues and organs of the body result in the occurrence of various senile diseases. Aging has been recognized as an important risk factor for cardiovascular disease. Endothelium is one of the most susceptible tissues, and endothelial cell senescence is the basis of endothelial senescence. It is of great significance to study the mechanism of endothelial cell senescence and to explore a new method to slow down the aging of endothelial cells for the prevention and treatment of cardiovascular disease. Free radical theory suggests that oxidative stress injury is one of the main causes of cell senescence. In this study, the aging model of human umbilical vein endothelial cells (HUVECs) was established by H _ 2O _ 2 (H _ 2O _ 2). The purpose of this study was to investigate the effect of hydrogen sulfide H _ 2S) on HUVECs senescence and its mechanism. Methods and results: 1. HUVECs were isolated by enzyme digestion and cultured in primary culture. The cells of the second generation of stable growth were selected. The cells were observed under inverted microscope. The cells were spindle-shaped, and the boundary was clear. The cells were cultured in vitro for 7 days. Cell fusion presents a typical paving stone arrangement. The expression of HUVECs specific marker factor 鈪

本文編號：1980411