miR-21靶向PDCD4影响的凋亡在造影剂所致急性肾损伤中的作用

发布时间：2018-04-23 01:07

本文选题：造影剂 + 急性肾损伤　；参考：《南方医科大学》2017年硕士论文

【摘要】：[研究背景]随着介入治疗的发展和增强CT的广泛运用,造影剂所致急性肾损伤(Contrast-induced acute kidney injury,CI-AKI)目前已成为医院获得性急性肾衰竭的第三大因素。CI-AKI的发生可增加患者身心和经济负担,同时对疾病远期预后产生负面影响。目前虽采用水化、限制造影剂剂量等方法来减少肾损伤,但高危人群中发生率仍可高达50%。因此,发病机制的研究显得尤为重要。近期研究表明,细胞凋亡是肾损伤的重要发病机制且与肾功能相关。因此,深入探讨造影剂诱导肾小管上皮细胞凋亡的分子机制,寻找关键靶点,将有利于CI-AKI的防治。miRNAs具有高度保守型和组织特异性,能稳定存在于体液和组织中,可通过抑制靶基因翻译或转录后表达在胚胎发育、细胞增殖分化和凋亡方面发挥多元化的调控作用。因此,miRNAs可成为基础和临床研究沟通的桥梁,在肿瘤领域中已是新型生物学标志物。近期发现,miRNAs同样可参与肾纤维化、缺血再灌注和肾毒性药物所致急性肾损伤过程。其中,miR-21在肾病中除存在差异性表达外,还同时参与调控细胞增殖、凋亡和纤维化过程,而此调控方向正是CI-AKI发生的重要部分。但不同疾病模型导致肾损伤机制不完全一致,因此miR-21表达和作用可能会有所差异。目前尚未有研究能明确miR-21在CI-AKI中的变化和调节作用。程序性细胞坏死因子4(Programmed cell death 4,PDCD4)是增殖细胞中可抑制蛋白转录和翻译过程的新型抑癌基因,同时也是近年来新发现的一种与细胞凋亡相关的基因。低表达水平的PDCD4可促进肿瘤细胞的增殖、侵袭和转移,而造影剂也可诱导肾小管上皮细胞发现凋亡和增殖,因此PDCD4可能参与调控CI-AKI的发生。另外,生物信息学分析软件提示miR-21与PDCD4存在互补碱基对。因此我们推测在CI-AKI中,miR-21可通过靶向PDCD4影响肾小管上皮细胞的凋亡。[研究目的]通过比较人肾小管上皮细胞(Human renal proximal tubular epithelial,HK-2)组和造影剂组中miR-21、PDCD4和凋亡指标,探讨造影剂对HK-2细胞凋亡和基因表达影响;采用荧光素酶报告,阐明miR-21对PDCD4的靶向调控;利用基因转染技术,探讨miR-21和PDCD4在该疾病模型中的作用。[研究方法](1)造影剂对miR-21和凋亡的影响:1.向HK-2中加入100mgI/ml、150mgI/ml和200mgI/ml碘普罗胺接触2h、2.5h和3h,观察细胞生长,检测细胞毒性和凋亡率;2.采用qRT-PCR检测miR-21和PDCD4,Western-blot检测凋亡相关蛋白和 PDCD4。(2)miR-21功能学研究:1.应用基因转染技术,向HK-2细胞内转染miR-21-mimics,miR-21-inhibitor 和 miR-21-NC,利用 qRT-PCR 检测 miR-21 和PDCD4;2.TUNEL染色观察组间细胞凋亡;3.Western-blot检测凋亡相关蛋白和PDCD4。(3)miR-21对PDCD4-3'UTR的靶向调控:1.生物学软件明确miR-21与PDCD4的配对碱基;2.合成野生型和突变型PDCD4,利用荧光素酶报告明确miR-21对PDCD4的靶向调节。(4)PDCD4在造影剂诱导细胞损伤中的作用:1.细胞内分别转染siRNA-PDCD4和siRNA-NC,qRT-PCR法检测PDCD4;2.Westem-blot检测凋亡相关蛋白和PDCD4。[研究结果](1)造影剂影响HK-2细胞状态:提升造影剂浓度和接触时间,细胞毒性和凋亡数目增加;显微镜下活动度下降、体积缩小、核质浓缩、细胞间连接减少。(2)造影剂诱导HK-2细胞凋亡,抑制miR-21表达:HK-2细胞接触造影剂后凋亡细胞数增多,Bcl-2蛋白量下降,Bax含量升高,miR-21呈低表达(P0.05)。(3)上调miR-21表达降低造影剂诱导HK-2细胞凋亡:细胞转染miR-21-mimics后,凋亡减少,Bcl-2蛋白量升高,Bax含量下降;转染miR-21-inhibitor后呈相反趋势(P0.05);转染空白载体上述指标较前无差异(P0.05)。(4)miR-21靶向调节PDCD4:1.miR-21与PDCD4存在互补碱基对;2.造影剂升高HK-2中PDCD4基因和蛋白含量(P0.05);3.上调miR-21降低PDCD4表达,下调miR-21升高其表达,两者变化趋势相反(P0.05);4.转染野生型-PDCD4可使荧光表达量较突变型-PDCD4显著下降(P0.001)。(5)PDCD4影响凋亡程度:细胞成功转染siRNA-PDCD4后,PDCD4基因和蛋白含量下降,Bax表达减少,Bcl-2含量增高,与造影剂组比较差异具有统计学意义(P0.05)。[研究结论]造影剂诱导HK-2细胞发生凋亡,升高PDCD4含量,抑制miR-21表达。miR-21-PDCD4-凋亡在造影剂诱导HK-2细胞损伤中发挥重要作用,为分子机制研究和后续在体验证奠定基础。
[Abstract]:[background] with the development of interventional therapy and the extensive use of CT, the acute renal injury (Contrast-induced acute kidney injury, CI-AKI) caused by contrast agent (CI-AKI) has become the third major factor of acute renal failure in hospital. The occurrence of.CI-AKI can increase the physical and mental and economic burden of the patients, and at the same time, it has a negative effect on the long-term prognosis of the disease. However, the incidence of renal injury in high risk population can still be as high as 50%., but the incidence of high risk population can still be as high as 50%.. The molecular mechanism of epithelial cell apoptosis and the search for key targets will be beneficial to the prevention and control of CI-AKI.MiRNAs with highly conserved and tissue specificity, which can be stable in body fluids and tissues, and can play a pluralistic role in the regulation of embryo development, cell proliferation and apoptosis by inhibiting target gene translation or post transcriptional expression. MiRNAs can be a bridge between basic and clinical research, which is a new biomarker in the field of cancer. Recently, miRNAs can also participate in the process of renal fibrosis, ischemia-reperfusion and renal toxicity induced acute renal injury. In addition, miR-21 is also involved in the regulation of cell proliferation and withering in kidney disease. The process of death and fibrosis is an important part of the occurrence of CI-AKI. However, the mechanisms of renal damage in different disease models are not completely consistent, so the expression and role of miR-21 may vary. There is no study to be able to identify the changes and regulation of miR-21 in CI-AKI. Death 4, PDCD4) is a new tumor suppressor gene that inhibits the transcription and translation of protein in proliferating cells. It is also a newly discovered gene related to apoptosis in recent years. The low expression level of PDCD4 can promote the proliferation, invasion and metastasis of tumor cells, and the contrast agent can also induce apoptosis and proliferation of renal tubular epithelial cells. Therefore, PDCD4 may be involved in the regulation of the occurrence of CI-AKI. In addition, bioinformatics analysis software suggests that miR-21 and PDCD4 have complementary base pairs. Therefore, we speculate that in CI-AKI, miR-21 can affect the apoptosis of renal tubular epithelial cells by targeting PDCD4. [Objective] by comparing human renal tubular epithelial cells (Human renal proximal tubular epit). Helial, HK-2) and contrast agent group, miR-21, PDCD4 and apoptosis index, explore the effect of contrast agent on the apoptosis and gene expression of HK-2 cells; use Luciferase Report, clarify the targeting regulation of miR-21 to PDCD4; explore the role of miR-21 and PDCD4 in the model of the disease by using gene transfection technique. [research method] (1) contrast agent on miR-21 and apoptosis 1. to add 100mgI/ml to HK-2, 150mgI/ml and 200mgI/ml iodiproamine contact 2h, 2.5h and 3h, observe cell growth, detect cytotoxicity and apoptosis rate; 2. use qRT-PCR to detect miR-21 and PDCD4, Western-blot detect apoptosis related proteins and PDCD4. (2) functional study: 1. application gene transfection technology, transfection into cells -21-mimics, miR-21-inhibitor and miR-21-NC, miR-21 and PDCD4 were detected by qRT-PCR; 2.TUNEL staining was used to observe the apoptosis between groups; 3.Western-blot detected apoptosis related proteins and PDCD4. (3) miR-21 to PDCD4-3'UTR targeting regulation: 1. biological software clearly defined the pairing of miR-21 with the base; 2. synthetic wild type and mutant type, utilization The luciferase report clearly regulates the targeting of miR-21 on PDCD4. (4) the role of PDCD4 in cell injury induced by contrast agents: siRNA-PDCD4 and siRNA-NC in 1. cells, PDCD4 in qRT-PCR; 2.Westem-blot detection of apoptosis related proteins and PDCD4.[study results] (1) the influence of the promoter on the state of HK-2 cells: enhancing the concentration and contact of contrast agents Time, the number of cytotoxicity and apoptosis increased; the activity of the microscope decreased, the volume reduced, the nuclear substance was concentrated, and the intercellular connection decreased. (2) the contrast agent induced the apoptosis of HK-2 cells and inhibited the expression of miR-21: the number of apoptotic cells, the decrease of Bcl-2 protein, the increase of Bax content and the low expression of Bax (P0.05). (3) up regulation of miR-21 Expression reduced contrast agent to induce apoptosis of HK-2 cells: after transfection of miR-21-mimics, apoptosis decreased, Bcl-2 protein increased and Bax content decreased; miR-21-inhibitor transfected after transfection was opposite (P0.05). The above indexes transfected with blank carrier (P0.05). (4) miR-21 targeting regulation PDCD4:1.miR-21 and PDCD4 existed complementary base pairs; 2. angiography The content of PDCD4 gene and protein content (P0.05) increased in HK-2; 3. up regulated miR-21 and lowered PDCD4 expression and lowered miR-21 to increase its expression. The change trend was opposite (P0.05); 4. transfection of wild type -PDCD4 could significantly decrease the fluorescence expression of -PDCD4 (P0.001). (5) PDCD4 affects the apoptosis degree: after successful transfection of siRNA-PDCD4 to cells, the gene and The protein content decreased, the expression of Bax decreased and the content of Bcl-2 increased, and the difference between the contrast agent group was statistically significant (P0.05). [Conclusion] the contrast agent induced the apoptosis of HK-2 cells and increased the content of PDCD4, and the inhibition of miR-21 expression.MiR-21-PDCD4- apoptosis played an important role in the injury of HK-2 cells induced by contrast agents. The follow-up lays the foundation for the experience.

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R541.4;R692.5

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