本文選題：蛋白結(jié)合的對(duì)甲酚　切入點(diǎn)：人臍靜脈內(nèi)皮細(xì)胞　出處：《第三軍醫(yī)大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：背景： 成人慢性腎臟�。–KD）的發(fā)病率高達(dá)10%[1]，其中相當(dāng)部分患者將發(fā)展到終末期腎衰，由于腎源奇缺、腎移植數(shù)量減少，絕大部分終末期腎衰患者只能靠透析生存。盡管近幾十年來，在透析技術(shù)、透析耗材、透析液等方面做了大量的研究和改進(jìn)，維持性血液透析患者的年死亡率仍高達(dá)10%，在這些死亡患者中，心、腦血管事件是最重要的死亡原因[2]。透析雖然緩解了多種毒素、水電解質(zhì)紊亂、酸堿失衡等致死因子對(duì)生命的直接威脅，但對(duì)尿毒癥時(shí)高發(fā)的心、腦血管病變本身并無明顯緩解，這不得不令我們重新審視現(xiàn)代透析的局限性。 以往研究表明，內(nèi)皮功能紊亂加速了慢性腎功能衰竭（CRF）患者的加速性動(dòng)脈硬化進(jìn)程。一些與血漿白蛋白結(jié)合的尿毒癥毒素，如硫酸吲哚酚、同型半胱氨酸、馬尿酸等，被證實(shí)參與損傷CKD患者的血管內(nèi)皮功能[3]。但仍難以解釋造成尿毒癥患者居高不下心腦血管并發(fā)癥的原因。 文獻(xiàn)表明，透析并不能清除蛋白結(jié)合的尿毒癥毒素。對(duì)甲酚，是一種與血漿白蛋白高度結(jié)合的尿毒癥毒素，與血漿白蛋白的結(jié)合率高達(dá)94%，常規(guī)的透析方法很難將其清除[4]。在CKD的中晚期階段，患者血清對(duì)甲酚水平明顯增高，同時(shí)與CKD患者的全因死亡率和心血管疾病發(fā)生率顯著相關(guān)[5]。近年來，蛋白結(jié)合毒素對(duì)甲酚在CKD患者心腦血管并發(fā)癥高發(fā)生率中所發(fā)揮的作用逐漸受到人們的關(guān)注。 目的： 本研究以培養(yǎng)的人臍靜脈內(nèi)皮細(xì)胞（HUVEC）為研究對(duì)象，觀察游離的對(duì)甲酚與蛋白結(jié)合的對(duì)甲酚（+4%白蛋白）對(duì)人臍靜脈內(nèi)皮細(xì)胞的增殖和細(xì)胞周期的影響及其機(jī)制，從而探討蛋白結(jié)合尿毒癥毒素對(duì)甲酚與CKD患者加速性動(dòng)脈硬化的關(guān)系。 方法： 本課題分為2部分進(jìn)行探討。 第一部分：觀察游離的對(duì)甲酚與蛋白結(jié)合的對(duì)甲酚對(duì)人臍靜脈內(nèi)皮細(xì)胞增殖、細(xì)胞形態(tài)、細(xì)胞凋亡和細(xì)胞周期的影響。 1.用CCK-8法觀察游離的對(duì)甲酚與蛋白結(jié)合的對(duì)甲酚對(duì)HUVEC細(xì)胞增殖的影響。 2.用倒置顯微鏡觀察對(duì)甲酚對(duì)HUVEC細(xì)胞形態(tài)的影響。 3.用細(xì)胞凋亡檢測(cè)試劑盒檢測(cè)對(duì)甲酚對(duì)人臍靜脈內(nèi)皮細(xì)胞凋亡的影響。 4.用流式細(xì)胞技術(shù)檢測(cè)對(duì)甲酚對(duì)人臍靜脈內(nèi)皮細(xì)胞周期的影響。 第二部分：觀察游離的對(duì)甲酚與蛋白結(jié)合的對(duì)甲酚對(duì)人臍靜脈內(nèi)皮細(xì)胞細(xì)胞周期調(diào)節(jié)蛋白的影響。 1.用Western印記法檢測(cè)對(duì)甲酚對(duì)對(duì)人臍靜脈內(nèi)皮細(xì)胞細(xì)胞周期調(diào)節(jié)蛋白p21Cip1,p27Kip1, p15Ink4B, p16Ink4A，CDK4，cyclin D1表達(dá)的影響。 2.用雙重免疫熒光法觀察對(duì)甲酚對(duì)人臍靜脈內(nèi)皮細(xì)胞周期調(diào)節(jié)蛋白p21Cip1和cyclin D1表達(dá)的影響。 結(jié)果： 第一部分： 1.游離的對(duì)甲酚與蛋白結(jié)合的對(duì)甲酚均抑制人臍靜脈內(nèi)皮細(xì)胞的增殖：與對(duì)照組相比，對(duì)甲酚80μg/ml組24h、48h、72h的細(xì)胞增殖抑制率分別為23.56%±3.24%、48.55%±5.05%、80.68%±1.69%。與對(duì)照+4%白蛋白組相比，80μg/ml對(duì)甲酚+4%白蛋白組24h、48h、72h的細(xì)胞增殖抑制率分別為22.39%±4.79%、41.38%±2.10%、77.56%±2.06%。且隨著對(duì)甲酚濃度的升高，其對(duì)細(xì)胞增殖的抑制作用逐漸增強(qiáng)（P0.05），同時(shí)隨著作用時(shí)間的延長(zhǎng)，這種抑制程度逐漸增強(qiáng)（P0.05）。游離的對(duì)甲酚與蛋白結(jié)合的對(duì)甲酚對(duì)HUVEC增殖的抑制作用均呈劑量依賴性和時(shí)間依賴性（P0.05）。 2.對(duì)甲酚處理后的HUVEC細(xì)胞形態(tài)發(fā)生了改變，呈現(xiàn)不規(guī)則狀，細(xì)胞突起伸長(zhǎng)，細(xì)胞質(zhì)變渾濁。 3.游離的對(duì)甲酚與蛋白結(jié)合的對(duì)甲酚對(duì)HUVEC細(xì)胞凋亡無影響（P0.05）。 4.游離的對(duì)甲酚與蛋白結(jié)合的對(duì)甲酚均使HUVEC停滯于細(xì)胞周期G1期：對(duì)照組G1期細(xì)胞比例為62.37±0.39%，S期細(xì)胞比例為28.96±1.04%；對(duì)甲酚80μg/ml組G1期細(xì)胞比例為81.27±1.15%，S期細(xì)胞比例為5.77±1.32%。對(duì)照+4%白蛋白組G1期細(xì)胞比例為62.45±0.72%，S期細(xì)胞比例為22.48±3.15%；對(duì)甲酚80μg/ml+4%白蛋白組G1期細(xì)胞比例為79.63±1.18%，S期細(xì)胞比例為8.86±0.21%。各濃度對(duì)甲酚組G1期細(xì)胞較對(duì)照組顯著增多（p0.05），S期細(xì)胞較對(duì)照組顯著減少（p0.05），且對(duì)甲酚所導(dǎo)致的G1期停滯呈劑量依賴性（p0.05）。 第二部分： 1.游離的對(duì)甲酚與蛋白結(jié)合的對(duì)甲酚均使HUVEC細(xì)胞周期調(diào)節(jié)蛋白p21Cip1表達(dá)增加（p0.05），cyclin D1蛋白表達(dá)減少（p0.05），且這種作用均呈劑量依賴性（p0.05）；而對(duì)細(xì)胞周期調(diào)節(jié)蛋白p27Kip1, p15Ink4B, p16Ink4A，CDK4表達(dá)無影響（P0.05）。 2.游離的對(duì)甲酚與蛋白結(jié)合的對(duì)甲酚增加了HUVEC細(xì)胞周期調(diào)節(jié)蛋白p21Cip1的表達(dá)，減少了cyclin D1蛋白的表達(dá)。 結(jié)論： 1.蛋白結(jié)合的對(duì)甲酚與游離對(duì)甲酚一樣，能顯著抑制HUVEC的增殖，，且呈劑量依賴性和時(shí)間依賴性。 2.蛋白結(jié)合的對(duì)甲酚與游離對(duì)甲酚對(duì)HUVEC增殖的抑制作用可能是通過上調(diào)細(xì)胞周期調(diào)節(jié)蛋白p21Cip1的表達(dá)，下調(diào)cyclin D1蛋白的表達(dá)，進(jìn)而使HUVEC停滯于細(xì)胞周期G1期所致，與細(xì)胞凋亡無關(guān)。
[Abstract]:Background:
Adult chronic kidney disease (CKD) incidence rate is as high as 10%[1], which is part of patients will develop to end-stage renal failure, because the kidneys are scarce, reduce the number of renal transplantation in patients with end-stage renal failure, most can only rely on dialysis to survive. Although in recent decades, in dialysis, dialysis supplies, such as dialysis fluid research and improvement of a large number of patients with maintenance hemodialysis mortality is still as high as 10%, in the death of patients, heart and cerebrovascular events is the most important cause of death in dialysis [2]. although alleviate a variety of toxins, water electrolyte disorder, acid-base imbalance of lethal direct threat to life, but for uremia the high incidence of heart cerebrovascular disease itself does not significantly alleviate the limitations, it had to make us re-examine modern dialysis.
Previous studies showed that endothelial dysfunction accelerates the chronic renal failure (CRF) accelerated the progression of atherosclerosis in patients with uremic toxins. Combined with plasma albumin, such as indoxyl sulfate, homocysteine, hippuric acid, was confirmed in [3]. vascular endothelial function injury in patients with CKD but it is still difficult to explain the cause of uremic patients the high cardiovascular complications.
The literature suggests that dialysis did not clear the uremic toxin protein binding. P-cresol is a uremic toxin and plasma albumin with up to 94%, and combined with the plasma albumin dialysis rate, the conventional method is difficult to remove [4]. in the late stage of CKD patients, serum p-cresol levels were significantly increased. At the same time in patients with CKD due to the incidence of [5]. was significantly associated with cardiovascular disease mortality in recent years, protein binding toxin paracresol in CKD patients with cardio cerebral vascular complications occurred in the high rate play a role gradually attracted people's attention.
Objective:
In this study, the cultured human umbilical vein endothelial cells (HUVEC) as the research object, observe the free combination of p-cresol and protein p-cresol (+4% albumin) influence the proliferation and cell cycle of human umbilical vein endothelial cells and its mechanism, and to explore the relationship of protein bound uremic toxin cresol and CKD in patients with accelerated artery hardening.
Method錛

本文編號(hào)：1647624