Sp1在高糖诱导肾小球足细胞凋亡中的作用及机制研究

发布时间：2018-10-24 11:13

【摘要】：研究背景:糖尿病肾病(Diabetic nephropathy,DN)是糖尿病病人常见的重要并发症之一,发病率在我国呈逐渐上升趋势,随着病情的进展而成为终末期肾脏病,因此,研究者们尽量想了解糖尿病肾病中肾小球损伤的机制,希望发现新的治疗策略。足细胞是高度终末分化的肾小球脏层上皮细胞,覆盖于肾小球基底膜(Glomerular basement membrane,GBM)外侧,在肾小球功能的调节中发挥着至关重要的作用。目前研究一致认为足细胞在DN的发病机制中发挥重要的作用。足细胞损伤表型改变的主要特征为细胞凋亡,体外和体内实验均证实高糖(HG)诱导足细胞凋亡,从而引起其损伤和数量的减少。但其具体机制目前仍然不甚清楚,因此,有必要对DN中足细胞凋亡所致的机制进一步深入研究,为临床预防和治疗DN提供理论依据。转录因子特异性蛋白-1(Sp1)属于SP/KLF转录因子家族成员之一,之前的研究数据显示:Sp1在人类包括甲状腺、胰腺、肝细胞、结直肠和胃癌各种肿瘤中高表达,这可能是肿瘤存在或发展的关键因素。最近,越来越多的证据表明Sp1与细胞凋亡有关,同时一些研究也证实Sp1还参与DN的发病机制。与此同时,有研究表明Sp1在维持足细胞完整性尤为重要,然而,HG是否激活Sp1在足细胞中的表达从而导致足细胞的凋亡还不清楚,本研究的目的是探讨高葡萄糖是否激活体外培养足细胞Sp1的表达以及其在高糖诱导足细胞凋亡中的可能机制。研究方法永生的小鼠足细胞在含有正常葡萄糖(NG)、高糖(HG)或Sp1 siRNA培养基中培养,用实时PCR、免疫印迹和免疫荧光测定足细胞中Sp1的表达。在使用Sp1 siRNA下调Sp1表达的情况下,通过流式细胞分析仪检测Sp1在高糖诱导足细胞凋亡中的作用。使用实时PCR、免疫印迹方法检测细胞凋亡基因Bax的mRNA和蛋白质的表达。统计分析采用SPSS17.0,数据用均值士标准差表示。多个组之间的比较使用单向方差分析与Bonferonni's/Turkey检验或Dunnett's T3检验。P0.05认为有统计学差异。结果:在培养的足细胞中,高糖以时间和剂量依赖的方式刺激Sp1的表达。用Sp1 siRNA预处理后(50nm)明显降低Sp1的表达;同时,高糖引起的细胞凋亡效应也被明显抑制。此外,我们还发现HG也增加了足细胞中Bax的表达,这一效应同样能被Sp1 siRNA所下调。结论:Sp1/Bax信号通路在HG诱导足细胞凋亡中发挥作用,阻断Sp1/Bax信号通路可以减少高糖诱导的足细胞凋亡。
[Abstract]:Background: diabetic nephropathy (Diabetic nephropathy,DN) is one of the most common complications in diabetic patients. Researchers try to understand the mechanism of glomerular damage in diabetic nephropathy and hope to find new treatment strategies. Podocytes are highly terminal differentiated epithelial cells in the visceral layer of the glomeruli, covering the outer side of the glomerular basement membrane (Glomerular basement membrane,GBM). Podocytes play an important role in the regulation of glomerular function. Current studies agree that podocytes play an important role in the pathogenesis of DN. Apoptosis is the main characteristic of podocyte damage phenotypic changes. In vitro and in vivo experiments confirmed that high glucose (HG) induced podocyte apoptosis, which resulted in the damage and decrease of the number of podocytes. However, its specific mechanism is still unclear, so it is necessary to further study the mechanism of podocyte apoptosis in DN, to provide a theoretical basis for clinical prevention and treatment of DN. Transcription factor-specific protein-1 (Sp1) is a member of the SP/KLF transcription factor family. Previous studies have shown that Sp1 is highly expressed in human thyroid, pancreas, hepatocytes, colorectal and gastric cancer. This may be a key factor in the existence or development of tumors. Recently, there has been increasing evidence that Sp1 is associated with apoptosis, and some studies have confirmed that Sp1 is also involved in the pathogenesis of DN. At the same time, studies have shown that Sp1 is particularly important in maintaining podocyte integrity. However, it is not clear whether HG activates the expression of Sp1 in podocytes, leading to podocyte apoptosis. The aim of this study was to investigate whether high glucose activates the expression of Sp1 in cultured podocytes and its possible mechanism in inducing apoptosis of podocytes. Methods the immortalized mouse podocytes were cultured in the medium containing normal glucose (NG), high glucose (HG) or Sp1 siRNA. The expression of Sp1 in podocytes was detected by real-time PCR, immunoblotting and immunofluorescence. When Sp1 siRNA was used to down-regulate the expression of Sp1, the role of Sp1 in the apoptosis of podocyte induced by high glucose was detected by flow cytometry. The expression of mRNA and protein of apoptotic gene Bax was detected by real time PCR, Western blotting. The statistical analysis is expressed by the mean standard deviation of SPSS17.0, data. Unidirectional ANOVA and Bonferonni's/Turkey test or Dunnett's test were used to compare the two groups. P05 was considered to have statistical difference. Results: in cultured podocytes, high glucose stimulated the expression of Sp1 in a time and dose dependent manner. After pretreatment with Sp1 siRNA (50nm), the expression of Sp1 was significantly decreased, and the apoptotic effect induced by high glucose was also significantly inhibited. In addition, we found that HG also increased the expression of Bax in podocytes, which was also down-regulated by Sp1 siRNA. Conclusion: Sp1/Bax signaling pathway plays an important role in podocyte apoptosis induced by HG, and blocking Sp1/Bax signaling pathway can reduce the apoptosis induced by high glucose.
【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R587.2;R692.9

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