發(fā)布時間：2018-06-08 00:15

  本文選題：南蛇藤素 + 血管緊張素II��； 參考：《第三軍醫(yī)大學》2017年碩士論文

【摘要】：研究背景血管平滑肌細胞(vascular smooth muscle cells,VSMCs)衰老與心血管疾病的發(fā)生發(fā)展緊密相關。細胞衰老的特征主要是氧化應激增加,端粒功能障礙和生長停滯,衰老相關蛋白β-半乳糖苷酶(senescence-associatedβ-galactosidase,SAβ-gal)、p53、p21和p16表達增加。血管緊張素II(angiotensin II,Ang II)在動脈粥樣硬化的發(fā)病機理中起重要作用,可增加活性氧(reactive oxygen species,ROS)的水平并降低細胞的抗氧化能力,最終通過p53/p21依賴機制導致血管衰老,因此,抑制Ang II活性可降低心血管疾病發(fā)病率和死亡率。越來越多的證據(jù)表明,使用某些藥物可以抑制或延緩細胞衰老的發(fā)生。已有的研究表明南蛇藤素(celastrol,CeT),這一來源于中藥雷公藤的五環(huán)三萜類化合物,具有抗氧化和促進細胞自噬的作用。內(nèi)皮祖細胞經(jīng)短時間的CeT預處理,即能顯著抑制氧化低密度脂蛋白誘導的細胞內(nèi)ROS水平的升高和細胞衰老。CeT是否影響VSMCs的衰老過程,目前尚未見報道。我們推測,CeT可能通過誘導細胞自噬,而降低Ang II誘導的VSMCs中ROS水平,進而發(fā)揮抗衰老作用。研究方法1.采用衰老檢測試劑盒對細胞中的SAβ-gal進行染色,Western blot檢測細胞內(nèi)p53和p21的表達水平,流式細胞術檢測細胞周期,探討CeT能否抑制Ang II誘導的VSMCs衰老。2.采用流式細胞技術和ROS檢測試劑盒測定VSMCs內(nèi)ROS水平,探討Ce T能否抑制Ang II誘導的VSMCs中ROS升高。3.選用免疫熒光技術與激光掃描共聚焦顯微鏡檢測細胞內(nèi)LC3并計數(shù),Western blot檢測LC3 II表達量的變化,探討CeT是否通過促進細胞自噬,抑制Ang II誘導的VSMCs中ROS升高和衰老的作用。4.Western blot檢測PI3K/Akt/m TOR信號通路相關蛋白的表達水平。結(jié)果1.Ce T可抑制Ang II誘導的VSMCs衰老:SAβ-gal染色結(jié)果可見,VSMCs經(jīng)100 nmol/L Ang II處理2天后,SAβ-gal染色陽性細胞數(shù)目明顯增加,而10 nmol/L,50 n M,100 nmol/L CeT預處理使上述衰老比例相對下降,50 nmol/L的作用最為顯著;Western blot結(jié)果可見,50 nmol/L CeT預處理,可抑制Ang II對p53和p21表達水平的促進作用;流式技術結(jié)果可見,采用50 nmol/L CeT預處理后再用Ang II處理,和單純用Ang II處理相比,G0/G1期的細胞數(shù)目相對下降,而S/G2期相對上升。2.50 nmol/L CeT可抑制Ang II誘導的VSMCs中ROS水平的升高。3.CeT可能通過促進細胞自噬,而抑制Ang II誘導的VSMCs中ROS的產(chǎn)生和細胞衰老:免疫熒光技術結(jié)果可見,50 nmol/L CeT可顯著促進VSMCs的自噬水平;Western blot檢測進一步表明,Ce T可促進VSMCs中LC3 II的表達。自噬抑制劑3-甲基腺嘌呤(3-methyladenine,3Ma)可減弱CeT的抗衰老作用,以及Ce T抑制ROS生成的作用。4.Ce T可能通過抑制PI3K/Akt/mTOR信號通路促進VSMCs自噬:Western blot檢測表明,Ang II可顯著促進PI3K/Akt/m TOR/p70s6k蛋白的表達及其磷酸化水平,而使用CeT預處理VSMCs后,Ang II的上述作用可被顯著抑制。結(jié)論CeT可通過促進細胞自噬,抑制Ang II誘導的VSMCs內(nèi)ROS水平的升高,進而抑制Ang II誘導的VSMCs衰老;這可能與CeT抑制PI3K/Akt/m TOR信號轉(zhuǎn)導途徑相關。
[Abstract]:Background vascular smooth muscle cells (VSMCs) senescence is closely related to the development of cardiovascular disease. Cell aging is characterized by increased oxidative stress, telomere dysfunction and stagnation of telomere dysfunction and growth, and senescence related protein beta galactosidase (senescence-associated beta -galactosidase, SA beta -gal), p53, p21 and The expression of p16 increases. Angiotensin II (angiotensin II, Ang II) plays an important role in the pathogenesis of atherosclerosis, which can increase the level of reactive oxygen (reactive oxygen species, ROS) and reduce the antioxidant capacity of cells, and eventually lead to vascular senescence through p53/p21 dependence mechanism. Therefore, inhibition of Ang activation can reduce cardiovascular disease. Disease incidence and mortality. More and more evidence suggests that the use of certain drugs can inhibit or delay the occurrence of cell senescence. Studies have shown that celastrol (CeT), which is derived from the five ring three terpenoids of Tripterygium wilfordii, has the effect of antioxidation and promoting cell autophagy. The endothelial progenitor cells (EPCs) have a short time of Ce T preconditioning, which can significantly inhibit the increase of intracellular ROS level induced by oxidized low density lipoprotein and whether cell aging.CeT affects the aging process of VSMCs, has not yet been reported. We speculate that CeT may reduce the ROS level in VSMCs induced by Ang II by inducing autophagy, and then exerts anti-aging effect. Research methods 1. SA beta -gal in cells was stained with the senescence detection kit. The expression level of p53 and p21 in cells was detected by Western blot, cell cycle was detected by flow cytometry, and whether CeT could inhibit VSMCs senescence of Ang II induced VSMCs senescence was determined by flow cytometry and ROS detection kit. The increase of.3. in ROS in VSMCs was determined by immunofluorescence and laser scanning confocal microscopy to detect the intracellular LC3 and to detect the changes in the expression of LC3 II by Western blot. The expression level of 1.Ce T inhibited the VSMCs senescence induced by Ang II: the results of SA beta -gal staining showed that the number of SA beta positive cells increased obviously after 2 days after VSMCs 100 nmol/L Ang II, and 10, 50, and 100 of the pretreatment to reduce the proportion of the senescence. Blot results can be seen that 50 nmol/L CeT preconditioning can inhibit the promotion of Ang II on p53 and p21 expression, and flow technique results can be seen that the number of cells in the period is relatively low compared with Ang II after 50 nmol/L CeT preprocessing. The increase of ROS level in VSMCs may promote autophagy by promoting cell autophagy, which inhibits the production of ROS in VSMCs induced VSMCs and cell senescence. The results of immunofluorescence technique can be seen that 50 nmol/L CeT can significantly promote the autophagy level of VSMCs. 3-methyladenine (3Ma) can weaken the anti-aging effect of CeT and the action of Ce T to inhibit the formation of ROS,.4.Ce T may promote VSMCs autophagy by inhibiting the PI3K/Akt/mTOR signaling pathway. The above effects of Ang II can be significantly inhibited. Conclusion CeT can inhibit the increase of ROS level in VSMCs induced by Ang II by promoting autophagy, which may inhibit Ang II induced VSMCs senescence, which may be related to CeT PI3K/Akt/m signal transduction pathway.
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R54

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