雷公藤甲素对阿霉素肾病大鼠足细胞凋亡的影响及其作用机制的研究

发布时间：2018-05-15 15:14

本文选题：雷公藤甲素 + Ntoch信号通路　；参考：《青岛大学》2017年硕士论文

【摘要】：目的:通过实验观察雷公藤甲素对阿霉素肾病(ADR)大鼠足细胞凋亡的影响并初步探讨雷公藤甲素减轻足细胞损伤的作用机制。方法:将45只雄性Wistar大鼠随机分为对照组(N)、阿霉素肾病模型组(ADR)及雷公藤干预组(T)。其中ADR组和T组大鼠经尾静脉一次性注射阿霉素6.5mg/kg构建阿霉素肾病大鼠模型,而N组大鼠则经尾静脉一次性注射同体积的生理盐水。模型建立后,T组给予雷公藤甲素(200ug/kg·d)干预8周。于2、4、6、8周分别检测24h尿蛋白定量(24h UTP),于8周末采血,观察各组大鼠的生化指标如血尿素氮(BUN)、肌酐(Scr)、胱抑素C(Cys C),并在光镜、电镜下观察肾组织学改变,应用TUNEL法检测足细胞凋亡,免疫组织化学染色检测肾母细胞瘤基因(WT-1)半定量计数足细胞,实时荧光定量聚合酶链反应(RT-PCR)检测足细胞标志物突触极蛋白Synaptopodin、凋亡相关因子p53及Notch1 m RNA的表达变化,蛋白质印记法(western)测定Synaptopodin、Notch1、Hes1的蛋白表达。结果:(1)ADR组各时间点24h尿蛋白定量、BUN、Scr以及Cys C指标均高于N组,差异有统计学意义(P0.05);而足细胞密度显著降低(P0.05),肾小球系膜区增宽,且系膜细胞增生;T组Scr、Cys C、24h尿蛋白定量低于ADR组(P0.05);足细胞密度明显增加(P0.05);同时观察到肾小球病理改变程度较ADR组减轻。(2)8周末时,ADR组Synaptopodin m RNA和蛋白表达降低,p53 m RNA表达升高,足细胞体积肿胀增大,凋亡足细胞数目比N组明显增多(P0.05);与ADR组相比,T组Synaptopodin m RNA和蛋白表达增加,p53 m RNA表达下降,凋亡足细胞数目较ADR组明显减少(P0.05)。(3)结合RT-PCR及western结果显示ADR组Notch1 m RNA和蛋白的表达以及Hes1蛋白的表达均高于N组(P0.05),T组Notch1 m RNA和蛋白的表达及Hes1蛋白的表达低于ADR组(P0.05)。结论:1.阿霉素肾病大鼠模型中存在足细胞损伤、凋亡;在大鼠足细胞凋亡数增加的同时Notch信号通路的相关指标Notch1、靶基因Hes1的表达量增加,而且凋亡相关因子p53的表达同样增加,而足细胞标志蛋白Synaptopodin表达减少;2.Notch信号通路可能通过激活其靶基因Hes,调节凋亡相关因子p53诱导足细胞的凋亡;3.应用雷公藤甲素干预后发现,足细胞凋亡数较阿霉素肾病模型组减少,而Notch信号通路的表达也相对下降;造成阿霉素肾病足细胞凋亡的原因可能与Notch信号通路过表达有关,而雷公藤甲素可能通过抑制Notch信号通路过表达从而减轻足细胞凋亡。
[Abstract]:Aim: to observe the effect of triptolide on apoptosis of podocyte in adriamycin nephropathy rats and to explore the mechanism of triptolide on podocyte injury. Methods: 45 male Wistar rats were randomly divided into control group, adriamycin nephropathy model group and tripterygium wilfordii intervention group. ADR group and T group rats were injected adriamycin 6.5mg/kg into caudal vein to establish the rat model of adriamycin nephropathy, while group N rats were injected with the same volume of normal saline via tail vein. After the model was established, T group was treated with triptolide (200ugkg / d) for 8 weeks. Blood samples were collected at the end of 8 weeks to observe the biochemical parameters such as blood urea nitrogen bun, creatinine cinnamon, cystatin C(Cys cleave, renal histologic changes under light microscope and electron microscope, and the apoptosis of podocyte by TUNEL method. Immunohistochemical staining was used to detect the semi-quantitative counting of podocytes, and real-time fluorescence quantitative polymerase chain reaction (RT-PCR- PCR) was used to detect the expression of Synaptopodin, apoptosis-related factor p53 and Notch1 m RNA. The protein expression of Synaptopodin Notch1 Hes1 was determined by Western blot. Results the levels of BUNC and Cys C in 24 h urinary protein quantitation in the 1 / 1 ADR group were significantly higher than those in N group (P 0.05), while the density of podocyte decreased significantly and the glomerular Mesangial area widened. In addition, the 24-hour urinary protein quantity of Scr-Cys Cnn in Mesangial cell proliferation T group was lower than that of ADR group (P 0.05), and the density of podocyte was significantly increased compared with that of ADR group. At the end of 8 weeks, the expression of Synaptopodin m RNA and protein in ADR group was decreased, and the expression of p53 m RNA was decreased. The number of apoptotic podocytes in podocyte was significantly increased than that in N group (P 0.05), the expression of Synaptopodin m RNA and protein in T group was higher than that in ADR group, and the expression of p53 m RNA was decreased in T group. The expression of Notch1 m RNA and protein and the expression of Hes1 protein in ADR group were significantly higher than those in group N (P 0.05). The expression of Notch1 m RNA and protein and the expression of Hes1 protein were significantly lower in ADR group than in ADR group (P 0.05). The number of apoptotic podocytes was significantly lower than that in ADR group. The results showed that the expression of Notch1 m RNA and protein and the expression of Hes1 protein in ADR group were significantly higher than those in N group. Conclusion 1. In the rat model of adriamycin nephropathy, podocytes were injured and apoptotic, and the expression of target gene Hes1 and apoptosis-related factor p53 was also increased when the number of apoptosis in podocyte increased and the related index of Notch signaling pathway Notch1 was increased, and the expression of apoptosis-related factor p53 was also increased in the rat model of adriamycin nephropathy. The decrease of Synaptopodin expression of podocyte marker protein 2. Notch signaling pathway may regulate apoptosis induced by apoptosis-related factor p53 by activating its target gene Hes3. After the intervention of triptolide, it was found that the number of podocyte apoptosis was lower than that of adriamycin nephropathy model group, and the expression of Notch signal pathway was also relatively decreased. The possible cause of podocyte apoptosis in doxorubicin nephropathy was the overexpression of Notch signal pathway. Triptolide may reduce podocyte apoptosis by inhibiting overexpression of Notch signaling pathway.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R692

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