蕓香苷對順鉑誘導(dǎo)人腎小球系膜細胞毒性的保護作用及機制研究
本文選題:蕓香苷 + 順鉑; 參考:《吉林大學(xué)》2017年碩士論文
【摘要】:背景:DDP是我國目前常用的一種化療藥物,對于多種惡性腫瘤具有明顯療效,但同時又伴有嚴重的毒副作用,其中以腎毒性最為明顯,嚴重影響了DDP的療效和臨床應(yīng)用。DDP誘發(fā)腎毒性的機制可能與氧自由基的堆積,凋亡因子的激活有關(guān)。目前減輕DDP腎毒方法是采用水化療法,但該療法易引發(fā)患者體內(nèi)電解質(zhì)紊亂,故仍存爭議。蕓香苷具有降低毛細血管通透性、抗炎、抗過敏、抗菌、抗病毒、抑制醛糖還原酶等活性。本實驗選擇HMCs作為研究對象,探究蕓香苷對DDP誘發(fā)腎毒性的保護作用及其相關(guān)機制。目的:通過檢測SOD、MDA、p53、pro-caspase-9、pro-caspase-3、cleavedcaspase-3、Bax及Bcl-2在HMCs中的表達情況,研究蕓香苷對DDP腎毒性的保護作用,并對其作用機制進行初步探討。方法:將細胞分為5組,即正常組(CON組)、模型組(MOD組)、蕓香苷50μM組(RUT 50μM組),蕓香苷25μM組(RUT 25μM組)、蕓香苷12.5μM組(RUT 12.5μM組)。應(yīng)用MTT、熒光染色、活性氧探針、流式細胞術(shù)、及Western Blotting等方法檢測HMCs的凋亡率、形態(tài)變化、活性氧含量以及SOD、MDA、p53、pro-caspase-9、pro-caspase-3、cleaved-caspase-3、Bax及Bcl-2在細胞中的表達情況。結(jié)果:1.MTT結(jié)果顯示,細胞培養(yǎng)24h后,DDP可以顯著降低細胞存活率,不同濃度蕓香苷均能提高細胞存活率;2.細胞形態(tài)學(xué)結(jié)果顯示,給藥24h后,MOD組死細胞增多,細胞發(fā)生分散、破碎;蕓香苷各劑量組與MOD組相比,死細胞減少,細胞形態(tài)明顯改善;3.流式細胞儀檢測結(jié)果顯示,給藥24h后,MOD組細胞出現(xiàn)明顯的周期阻滯,凋亡細胞數(shù)增多;與MOD組相比,蕓香苷25μM、12.5μM劑量組細胞周期阻滯得到改善,凋亡細胞數(shù)減少;4.活性氧含量及SOD、MDA檢測結(jié)果顯示,給藥24h后,MOD組細胞內(nèi)活性氧含量增加,SOD活力降低,MDA含量升高;蕓香苷各劑量組與MOD組相比,細胞內(nèi)活性氧含量降低,SOD活力升高,MDA含量降低;5.Western Blotting結(jié)果顯示,MOD組細胞p53、cleaved-caspase-3蛋白表達水平顯著提高,而pro-caspase-9、pro-caspase-3及Bcl-2/Bax蛋白水平明顯降低;與MOD組相比,蕓香苷各劑量組可降低細胞內(nèi)p53、cleaved-caspase-3蛋白表達,上調(diào)pro-caspase-9、pro-caspase-3及Bcl-2/Bax蛋白表達,尤以25μM組效果最佳。結(jié)論:1.蕓香苷能夠提高HMCs細胞存活率、改善細胞形態(tài)和周期阻滯,并可降低細胞內(nèi)ROS和MDA的含量,提高SOD活力,說明蕓香苷對DDP誘發(fā)HMCs毒性具有保護作用;2.蕓香苷可顯著降低凋亡細胞數(shù),其可能機制是通過抑制細胞內(nèi)p53、cleaved-caspase-3蛋白表達,上調(diào)pro-caspase-9、pro-caspase-3及Bcl-2/Bax蛋白表達來實現(xiàn)的,由此推斷蕓香苷可通過抑制p53/caspase信號通路中相關(guān)凋亡因子的表達實現(xiàn)其對腎臟的保護作用。
[Abstract]:Background: DDP is a commonly used chemotherapeutic drug in China at present. It has obvious curative effect on many kinds of malignant tumors, but it also has serious side effects, among which nephrotoxicity is the most obvious. The mechanism of renal toxicity induced by DDP may be related to the accumulation of oxygen free radicals and the activation of apoptosis factors. At present, hydration therapy is used to alleviate DDP nephrotoxicity, but this therapy is prone to lead to electrolyte disturbance in patients, so it is still controversial. Rutin has decreased capillary permeability, anti-inflammatory, anti-allergic, antibacterial, anti-virus, inhibition of Aldose reductase and other activities. In this study, HMCs were selected to investigate the protective effect of rutin on DDP induced nephrotoxicity and its related mechanisms. Objective: to study the protective effect of rutin on renal toxicity of DDP by detecting the expression of Bax and Bcl-2 in HMCs by detecting the expression of pro-caspase-9, pro-caspase-9 and pro-caspase-9 in HMCs. Methods: the cells were divided into five groups: normal group (Con), model group (mod), rutin (50 渭 M), rut (25 渭 M), rut (12.5 渭 M) and rut (12.5 渭 M). MTT, fluorescence staining, reactive oxygen species probe, flow cytometry and Western blotting were used to detect the apoptosis rate, morphological changes, reactive oxygen species content and the expression of Bax and Bcl-2 in HMCs. Results: 1. MTT results showed that DDP could significantly decrease cell viability after 24 hours of cell culture, and different concentrations of rutin could increase cell survival. The results of cell morphology showed that after 24 hours of administration, the dead cells increased, the cells were dispersed and broken in the mod group, and the dead cells decreased and the cell morphology improved significantly in each dose of rutin group compared with the MOD group. The results of flow cytometry showed that the cell cycle arrest was obvious and the number of apoptotic cells was increased in the mod group 24 hours after administration, and the cell cycle arrest was improved and the number of apoptotic cells decreased by 4% in the 25 渭 M and 12.5 渭 M group compared with the MOD group. The results of reactive oxygen species (Ros) content and SOD MDA content in the cells after 24 hours of administration showed that the activity of SOD was increased and the MDA content was decreased after 24 h administration of Rutanyl, compared with that in the MOD group, and that in the different doses of rutin group was higher than that in the MOD group. Results of Western blotting showed that the expression of p53 cleaved-caspase-3 protein was significantly increased, while pro-caspase-9 pro-caspase-3 and Bcl-2 / Bax protein levels were significantly decreased in mod group, compared with those in MOD group. The expression of p53 cleaved-caspase-3 was decreased and pro-caspase-9 pro-caspase-3 and Bcl-2 / Bax protein were up-regulated in different dosage groups of rutin, especially in 25 渭 M group. Conclusion 1. Rutin could increase the survival rate of HMCs cells, improve cell morphology and cell cycle arrest, decrease the contents of Ros and MDA, and increase the activity of SOD, indicating that rutin has a protective effect on DDP induced HMCs toxicity. Rutin can significantly reduce the number of apoptotic cells, which may be achieved by inhibiting the expression of p53 cleaved-caspase-3 protein and up-regulating the expression of pro-caspase-9 pro-caspase-3 and Bcl-2 / Bax protein. It is concluded that rutin can protect kidney by inhibiting the expression of apoptotic factors in p53/caspase signaling pathway.
【學(xué)位授予單位】:吉林大學(xué)
【學(xué)位級別】:碩士
【學(xué)位授予年份】:2017
【分類號】:R285
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