本文選題：辛伐他汀 + 川芎嗪��； 參考：《南京中醫(yī)藥大學(xué)》2015年碩士論文

【摘要】：背景腹膜透析(Peritoneal dialysis, PD)為腎臟替代治療的一種方式,有較好地保護(hù)殘余腎功能、中分子物質(zhì)濾過效果好、可在家自行治療等優(yōu)點(diǎn),近年來成為部分終末期腎衰竭患者的首選腎臟替代治療方式。但隨著腹膜透析的持續(xù)進(jìn)行,腹膜纖維化(peritoneal fibrosis, PF)逐漸出現(xiàn)并日漸加重,為腹膜透析常見并發(fā)癥,其影響代謝廢物及水分的超濾,致使肌酐、尿素、胍類等毒素的清除效率下降,患者最終選擇退出腹膜透析,改行其他替代治療。在腹膜纖維化發(fā)生時(shí),腹膜中細(xì)胞外基質(zhì)(extracellular matrix, ECM)大量沉積,腹膜血管過度增生,腹膜間皮細(xì)胞損傷,纖維化逐漸加重。截止目前的研究已表明,高糖、低PH值、高滲透壓的腹膜透析液及其中的葡萄糖降解產(chǎn)物長期作用于腹膜以及反復(fù)發(fā)作的腹膜透析相關(guān)腹膜炎均可誘導(dǎo)致纖維化細(xì)胞因子如TGF-β、FGF、VEGF等的分泌,細(xì)胞外基質(zhì)大量沉積,血管增生,腹膜纖維化出現(xiàn)及加重。既往研究表明,組織型纖溶酶原激活物(t-PA)、纖溶酶原激活物抑制因子-1(PAI-1)是人體正常生命活動(dòng)所必需的細(xì)胞因子,調(diào)節(jié)體內(nèi)的纖溶過程,同時(shí)其分泌及表達(dá)在腹膜纖維化過程中也有重要影響,可干預(yù)腹膜纖維化的發(fā)生、發(fā)展。目的 該研究通過觀察川芎嗪和辛伐他汀對(duì)高糖誘導(dǎo)后的人腹膜間皮細(xì)胞(human peritoneal mesothelial cells, HPMCs)中t-PA、PAI-1的分泌及表達(dá),進(jìn)一步探討上述兩種藥物對(duì)上述指標(biāo)變化的影響響機(jī)制,為腹膜纖維化的預(yù)防及治療提供理論依據(jù)。方法所用人體腹膜來目鼓樓醫(yī)院腹部手術(shù)(非尿毒癥、腹膜炎)患者,經(jīng)胰蛋白酶消化、分離后得到腹膜間皮細(xì)胞,再進(jìn)行原代培養(yǎng)、傳代培養(yǎng),第三代細(xì)胞用于本實(shí)驗(yàn),各次實(shí)驗(yàn)均使用3名患者的腹膜間皮細(xì)胞實(shí)施3次獨(dú)立實(shí)驗(yàn)。用培養(yǎng)液同步48h后,將HPMCs分為8組(每組設(shè)3個(gè)樣木)觀察：空白對(duì)照組(N組,完全培養(yǎng)液),高糖誘導(dǎo)組(H組,2.5%葡萄糖),低劑量辛伐他汀組(高糖+2.5μmol/L辛伐他汀)、中劑量辛伐他汀組(S2組,高糖+5.0μmol/L辛伐他汀)、高劑量辛伐他汀組(S3組,高糖+10.0μmol/L辛伐他汀)、低劑量川芎嗪組(T1組,高糖+川芎嗪10mg/L)、中劑量川芎嗪組(T2組,高糖+川芎嗪20mg/L)、高劑量川芎嗪組(T3組,高糖+川芎嗪40mg/L)。MTT法檢測各組HPMCs的活性,RT-PCR法檢測細(xì)胞內(nèi)t-PA和PAI-1的表達(dá),ELISA法檢測細(xì)胞上清液中t-PA和PAI-1蛋白質(zhì)水平,細(xì)胞部分用BCA蛋白檢測法測定細(xì)胞蛋白質(zhì)表達(dá)量,以此校正ELISA結(jié)果。結(jié)果① 高糖誘導(dǎo)組和空白組比較,高濃度葡萄糖能明顯降低腹膜間皮細(xì)胞活性(P0.01),同時(shí)腹膜間皮細(xì)胞中t-PA生成減少和PAI-1的表達(dá)增強(qiáng)(P0.01)；②與高糖誘導(dǎo)組比較,辛伐他汀干預(yù)組腹膜間皮細(xì)胞的活性明顯升高(P0.01), t-PA生成增加、PAI-1生成顯著減少(P0.01), t-PA mRNA表達(dá)水平顯著增加、PAI-1 mRNA表達(dá)水平顯著降低(P0.01),在蛋白及基因水平上,兩者均與辛伐他汀存在量效關(guān)系;③與高糖誘導(dǎo)組比較,川芎嗪干預(yù)組腹膜間皮細(xì)胞活性明顯升高(P0.01),川芎嗪各干預(yù)組(T1、T2、T3組)腹膜間皮細(xì)胞活性明顯升高(P0.01), T1、T2、T3組中t-PA的表達(dá)量增加,PAI-1的表達(dá)量均明顯降低(P0.01),t-PA mRNA表達(dá)均明顯上調(diào)、PAI-1 mRNA表達(dá)明顯降低(P0.01),在蛋白和基因水平上,兩者也與川芎嗪有量效關(guān)系。結(jié)論辛伐他汀和川芎嗪能抑制腹膜間皮細(xì)胞中PAI-1的表達(dá),促進(jìn)t-PA的表達(dá),因此可減少細(xì)胞外基質(zhì)的大量積聚,有預(yù)防、抑制腹膜纖維化的作用。
[Abstract]:Peritoneal dialysis (PD) is a way of renal replacement therapy. It has the advantages of better protection of residual kidney function, good filtration effect of medium molecular material and self treatment at home. In recent years, it has become the first choice for renal replacement therapy in patients with partial end-stage renal failure. However, peritoneal dialysis continues to carry out peritoneum. Peritoneal fibrosis (PF) is gradually appearing and aggravating gradually. It is a common complication of peritoneal dialysis, which affects the ultrafiltration of metabolic waste and water. The removal efficiency of creatinine, urea, guanidine and other toxins is decreased. The patients choose to withdraw from peritoneal dialysis and change to other alternative treatments. The matrix (extracellular matrix, ECM) deposits, peritoneal hyperproliferation, peritoneal mesothelial cell damage, and fibrosis progressively. The current study has shown that high glucose, low pH, hypertonic Liquor Dialysisintraperitoneus and glucose degradation products are used for peritoneum and recurrent peritoneal dialysis related peritonitis for a long time. The secretion of fibrotic cytokines such as TGF- beta, FGF, VEGF and so on, a large amount of extracellular matrix deposition, vascular proliferation, and peritoneal fibrosis appear and aggravate. Previous studies have shown that tissue plasminogen activator (t-PA) and plasminogen activator inhibitor -1 (PAI-1) are essential cytokines in human normal life activities, regulatory bodies. The internal fibrinolysis process, and its secretion and expression in the process of peritoneal fibrosis also have important effects, can interfere with the occurrence of peritoneal fibrosis, development. Objective the purpose of this study through the observation of Tetramethylpyrazine and simvastatin on high glucose induced human peritoneal mesothelial cells (human peritoneal mesothelial cells, HPMCs), t-PA, PAI-1 secretion and expression, Further explore the effect of the above two drugs on the changes of the above indexes, and provide a theoretical basis for the prevention and treatment of peritoneal fibrosis. Methods the human peritoneum in the abdomen of the Drum Tower Hospital (non uremia, peritonitis), the peritoneal mesothelial cells were obtained after trypsin digestion, then the primary culture was carried out and the passage was passed. Culture, third generation of cells used in this experiment, each experiment used 3 patients with peritoneal mesothelial cells to carry out 3 independent experiments. After using the culture fluid to synchronize 48h, HPMCs was divided into 8 groups (each set of 3 samples): blank control group (group N, complete culture fluid), high glucose induction group (H group, 2.5% glucose), low dose simvastatin group (high glucose +2.5 Mu Mo). L/L simvastatin), medium dose simvastatin group (group S2, high glucose +5.0 mol/L simvastatin), high dose simvastatin group (S3 group, high glucose +10.0 mol/L simvastatin), low dose Ligustrazine group (T1 group, high sugar + Ligustrazine 10mg/L), middle dose Ligustrazine group (T2 group, high sugar + Ligustrazine 20mg/L), high dose Ligustrazine group (T3 group, high sugar + Ligustrazine) The activity of HPMCs in each group was detected by TT. The expression of t-PA and PAI-1 in cell was detected by RT-PCR method. The protein level of t-PA and PAI-1 in the cell supernatant was detected by ELISA method. The cell protein expression was measured by the BCA protein detection method. The results were corrected for the ELISA results. The activity of peritoneal mesothelial cells (P0.01) decreased and the expression of t-PA in peritoneal mesothelial cells decreased and the expression of PAI-1 increased (P0.01). Compared with the high glucose induction group, the activity of peritoneal mesothelial cells in the simvastatin group increased significantly (P0.01), the formation of t-PA was increased, the formation of PAI-1 was significantly reduced (P0.01), and the expression level of t-PA mRNA increased significantly, PAI-1. PAI-1 The expression level of mRNA was significantly decreased (P0.01), both in protein and gene level, both of which were related with simvastatin. Compared with the high glucose induction group, the activity of peritoneal mesothelial cells in the tetramethylpyrazine intervention group was significantly increased (P0.01). The activity of peritoneal mesothelial cells in the tetramethylpyrazine intervention group (T1, T2, T3 group) was significantly increased (P0.01), T1, T2, T3 group t-PA. The expression of PAI-1 decreased significantly (P0.01), the expression of t-PA mRNA was obviously up-regulated, and the expression of PAI-1 mRNA decreased significantly (P0.01). In protein and gene levels, both of them also had a quantitative relationship with Ligustrazine. Conclusion simvastatin and ligustrazine could inhibit the expression of PAI-1 in the peritoneal mesothelial cells and promote the expression of t-PA, thus reducing the expression of t-PA. The accumulation of extracellular matrix can prevent and inhibit peritoneal fibrosis.

【學(xué)位授予單位】：南京中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R692.5

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