芸香苷对顺铂诱导人肾小球系膜细胞毒性的保护作用及机制研究

发布时间：2018-06-06 15:39

本文选题：芸香苷 + 顺铂　；参考：《吉林大学》2017年硕士论文

【摘要】：背景:DDP是我国目前常用的一种化疗药物,对于多种恶性肿瘤具有明显疗效,但同时又伴有严重的毒副作用,其中以肾毒性最为明显,严重影响了DDP的疗效和临床应用。DDP诱发肾毒性的机制可能与氧自由基的堆积,凋亡因子的激活有关。目前减轻DDP肾毒方法是采用水化疗法,但该疗法易引发患者体内电解质紊乱,故仍存争议。芸香苷具有降低毛细血管通透性、抗炎、抗过敏、抗菌、抗病毒、抑制醛糖还原酶等活性。本实验选择HMCs作为研究对象,探究芸香苷对DDP诱发肾毒性的保护作用及其相关机制。目的:通过检测SOD、MDA、p53、pro-caspase-9、pro-caspase-3、cleavedcaspase-3、Bax及Bcl-2在HMCs中的表达情况,研究芸香苷对DDP肾毒性的保护作用,并对其作用机制进行初步探讨。方法:将细胞分为5组,即正常组(CON组)、模型组(MOD组)、芸香苷50μM组(RUT 50μM组),芸香苷25μM组(RUT 25μM组)、芸香苷12.5μM组(RUT 12.5μM组)。应用MTT、荧光染色、活性氧探针、流式细胞术、及Western Blotting等方法检测HMCs的凋亡率、形态变化、活性氧含量以及SOD、MDA、p53、pro-caspase-9、pro-caspase-3、cleaved-caspase-3、Bax及Bcl-2在细胞中的表达情况。结果:1.MTT结果显示,细胞培养24h后,DDP可以显著降低细胞存活率,不同浓度芸香苷均能提高细胞存活率;2.细胞形态学结果显示,给药24h后,MOD组死细胞增多,细胞发生分散、破碎;芸香苷各剂量组与MOD组相比,死细胞减少,细胞形态明显改善;3.流式细胞仪检测结果显示,给药24h后,MOD组细胞出现明显的周期阻滞,凋亡细胞数增多;与MOD组相比,芸香苷25μM、12.5μM剂量组细胞周期阻滞得到改善,凋亡细胞数减少;4.活性氧含量及SOD、MDA检测结果显示,给药24h后,MOD组细胞内活性氧含量增加,SOD活力降低,MDA含量升高;芸香苷各剂量组与MOD组相比,细胞内活性氧含量降低,SOD活力升高,MDA含量降低;5.Western Blotting结果显示,MOD组细胞p53、cleaved-caspase-3蛋白表达水平显著提高,而pro-caspase-9、pro-caspase-3及Bcl-2/Bax蛋白水平明显降低;与MOD组相比,芸香苷各剂量组可降低细胞内p53、cleaved-caspase-3蛋白表达,上调pro-caspase-9、pro-caspase-3及Bcl-2/Bax蛋白表达,尤以25μM组效果最佳。结论:1.芸香苷能够提高HMCs细胞存活率、改善细胞形态和周期阻滞,并可降低细胞内ROS和MDA的含量,提高SOD活力,说明芸香苷对DDP诱发HMCs毒性具有保护作用;2.芸香苷可显著降低凋亡细胞数,其可能机制是通过抑制细胞内p53、cleaved-caspase-3蛋白表达,上调pro-caspase-9、pro-caspase-3及Bcl-2/Bax蛋白表达来实现的,由此推断芸香苷可通过抑制p53/caspase信号通路中相关凋亡因子的表达实现其对肾脏的保护作用。
[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.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285

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