本文選題：造影劑 + 急性腎損傷�。� 參考：《南方醫(yī)科大學(xué)》2017年碩士論文

【摘要】：[研究背景]隨著介入治療的發(fā)展和增強(qiáng)CT的廣泛運(yùn)用,造影劑所致急性腎損傷(Contrast-induced acute kidney injury,CI-AKI)目前已成為醫(yī)院獲得性急性腎衰竭的第三大因素。CI-AKI的發(fā)生可增加患者身心和經(jīng)濟(jì)負(fù)擔(dān),同時(shí)對疾病遠(yuǎn)期預(yù)后產(chǎn)生負(fù)面影響。目前雖采用水化、限制造影劑劑量等方法來減少腎損傷,但高危人群中發(fā)生率仍可高達(dá)50%。因此,發(fā)病機(jī)制的研究顯得尤為重要。近期研究表明,細(xì)胞凋亡是腎損傷的重要發(fā)病機(jī)制且與腎功能相關(guān)。因此,深入探討造影劑誘導(dǎo)腎小管上皮細(xì)胞凋亡的分子機(jī)制,尋找關(guān)鍵靶點(diǎn),將有利于CI-AKI的防治。miRNAs具有高度保守型和組織特異性,能穩(wěn)定存在于體液和組織中,可通過抑制靶基因翻譯或轉(zhuǎn)錄后表達(dá)在胚胎發(fā)育、細(xì)胞增殖分化和凋亡方面發(fā)揮多元化的調(diào)控作用。因此,miRNAs可成為基礎(chǔ)和臨床研究溝通的橋梁,在腫瘤領(lǐng)域中已是新型生物學(xué)標(biāo)志物。近期發(fā)現(xiàn),miRNAs同樣可參與腎纖維化、缺血再灌注和腎毒性藥物所致急性腎損傷過程。其中,miR-21在腎病中除存在差異性表達(dá)外,還同時(shí)參與調(diào)控細(xì)胞增殖、凋亡和纖維化過程,而此調(diào)控方向正是CI-AKI發(fā)生的重要部分。但不同疾病模型導(dǎo)致腎損傷機(jī)制不完全一致,因此miR-21表達(dá)和作用可能會有所差異。目前尚未有研究能明確miR-21在CI-AKI中的變化和調(diào)節(jié)作用。程序性細(xì)胞壞死因子4(Programmed cell death 4,PDCD4)是增殖細(xì)胞中可抑制蛋白轉(zhuǎn)錄和翻譯過程的新型抑癌基因,同時(shí)也是近年來新發(fā)現(xiàn)的一種與細(xì)胞凋亡相關(guān)的基因。低表達(dá)水平的PDCD4可促進(jìn)腫瘤細(xì)胞的增殖、侵襲和轉(zhuǎn)移,而造影劑也可誘導(dǎo)腎小管上皮細(xì)胞發(fā)現(xiàn)凋亡和增殖,因此PDCD4可能參與調(diào)控CI-AKI的發(fā)生。另外,生物信息學(xué)分析軟件提示miR-21與PDCD4存在互補(bǔ)堿基對。因此我們推測在CI-AKI中,miR-21可通過靶向PDCD4影響腎小管上皮細(xì)胞的凋亡。[研究目的]通過比較人腎小管上皮細(xì)胞(Human renal proximal tubular epithelial,HK-2)組和造影劑組中miR-21、PDCD4和凋亡指標(biāo),探討造影劑對HK-2細(xì)胞凋亡和基因表達(dá)影響;采用熒光素酶報(bào)告,闡明miR-21對PDCD4的靶向調(diào)控;利用基因轉(zhuǎn)染技術(shù),探討miR-21和PDCD4在該疾病模型中的作用。[研究方法](1)造影劑對miR-21和凋亡的影響:1.向HK-2中加入100mgI/ml、150mgI/ml和200mgI/ml碘普羅胺接觸2h、2.5h和3h,觀察細(xì)胞生長,檢測細(xì)胞毒性和凋亡率;2.采用qRT-PCR檢測miR-21和PDCD4,Western-blot檢測凋亡相關(guān)蛋白和 PDCD4。(2)miR-21功能學(xué)研究:1.應(yīng)用基因轉(zhuǎn)染技術(shù),向HK-2細(xì)胞內(nèi)轉(zhuǎn)染miR-21-mimics,miR-21-inhibitor 和 miR-21-NC,利用 qRT-PCR 檢測 miR-21 和PDCD4;2.TUNEL染色觀察組間細(xì)胞凋亡;3.Western-blot檢測凋亡相關(guān)蛋白和PDCD4。(3)miR-21對PDCD4-3'UTR的靶向調(diào)控:1.生物學(xué)軟件明確miR-21與PDCD4的配對堿基;2.合成野生型和突變型PDCD4,利用熒光素酶報(bào)告明確miR-21對PDCD4的靶向調(diào)節(jié)。(4)PDCD4在造影劑誘導(dǎo)細(xì)胞損傷中的作用:1.細(xì)胞內(nèi)分別轉(zhuǎn)染siRNA-PDCD4和siRNA-NC,qRT-PCR法檢測PDCD4;2.Westem-blot檢測凋亡相關(guān)蛋白和PDCD4。[研究結(jié)果](1)造影劑影響HK-2細(xì)胞狀態(tài):提升造影劑濃度和接觸時(shí)間,細(xì)胞毒性和凋亡數(shù)目增加;顯微鏡下活動(dòng)度下降、體積縮小、核質(zhì)濃縮、細(xì)胞間連接減少。(2)造影劑誘導(dǎo)HK-2細(xì)胞凋亡,抑制miR-21表達(dá):HK-2細(xì)胞接觸造影劑后凋亡細(xì)胞數(shù)增多,Bcl-2蛋白量下降,Bax含量升高,miR-21呈低表達(dá)(P0.05)。(3)上調(diào)miR-21表達(dá)降低造影劑誘導(dǎo)HK-2細(xì)胞凋亡:細(xì)胞轉(zhuǎn)染miR-21-mimics后,凋亡減少,Bcl-2蛋白量升高,Bax含量下降;轉(zhuǎn)染miR-21-inhibitor后呈相反趨勢(P0.05);轉(zhuǎn)染空白載體上述指標(biāo)較前無差異(P0.05)。(4)miR-21靶向調(diào)節(jié)PDCD4:1.miR-21與PDCD4存在互補(bǔ)堿基對;2.造影劑升高HK-2中PDCD4基因和蛋白含量(P0.05);3.上調(diào)miR-21降低PDCD4表達(dá),下調(diào)miR-21升高其表達(dá),兩者變化趨勢相反(P0.05);4.轉(zhuǎn)染野生型-PDCD4可使熒光表達(dá)量較突變型-PDCD4顯著下降(P0.001)。(5)PDCD4影響凋亡程度:細(xì)胞成功轉(zhuǎn)染siRNA-PDCD4后,PDCD4基因和蛋白含量下降,Bax表達(dá)減少,Bcl-2含量增高,與造影劑組比較差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。[研究結(jié)論]造影劑誘導(dǎo)HK-2細(xì)胞發(fā)生凋亡,升高PDCD4含量,抑制miR-21表達(dá)。miR-21-PDCD4-凋亡在造影劑誘導(dǎo)HK-2細(xì)胞損傷中發(fā)揮重要作用,為分子機(jī)制研究和后續(xù)在體驗(yàn)證奠定基礎(chǔ)。
[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.

【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R541.4;R692.5

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相關(guān)期刊論文 前1條

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