本文選題：對(duì)比劑誘導(dǎo)的急性腎損傷 + 內(nèi)質(zhì)網(wǎng)應(yīng)激　； 參考：《天津醫(yī)科大學(xué)》2016年博士論文

【摘要】：研究背景及目的:對(duì)比劑誘導(dǎo)的急性腎損傷(contrast-induced acute kidney injury,CIAKI),又被稱之為對(duì)比劑腎病(contrast induced nephropathy,CIN),是應(yīng)用對(duì)比劑后發(fā)生的重要的臨床并發(fā)癥,它是行經(jīng)皮冠狀動(dòng)脈介入治療后繼支架內(nèi)血栓、支架內(nèi)再狹窄后的第三大并發(fā)癥。臨床應(yīng)用的對(duì)比劑主要包括泛影葡胺、碘佛醇、碘海醇等。CIAKI的發(fā)病機(jī)制十分繁雜。有研究表明,在CIAKI中腎小管損傷中,內(nèi)質(zhì)網(wǎng)應(yīng)激而誘導(dǎo)的細(xì)胞凋亡發(fā)揮重要作用。已有研究推測(cè),阿托伐他汀、普羅布考與前列地爾藥物的抗氧化與抗炎或改善腎血管灌注作用可抑制腎小管細(xì)胞凋亡,均可能預(yù)防CIAKI的發(fā)生。此前已有研究顯示阿托伐他汀可成功抑制內(nèi)質(zhì)網(wǎng)應(yīng)激誘導(dǎo)的細(xì)胞凋亡作用從而預(yù)防CIAKI的發(fā)生。然而,該聯(lián)合用藥是否可通過(guò)抑制內(nèi)質(zhì)網(wǎng)應(yīng)激誘導(dǎo)的細(xì)胞凋亡作用進(jìn)而預(yù)防CIAKI的發(fā)生尚無(wú)具體研究。近幾十年的研究發(fā)現(xiàn)許多可檢測(cè)CIAKI的早期新型標(biāo)記物,此前已有研究初步證明叢生蛋白可預(yù)測(cè)CIAKI的發(fā)生,但缺乏進(jìn)一步的論證。因此,本研究擬用泛影葡胺建立大鼠CIAKI模型,通過(guò)不同藥物聯(lián)合干預(yù),比較藥物聯(lián)合與阿托伐他汀單藥對(duì)大鼠對(duì)比劑腎損傷的影響,檢測(cè)各種藥物聯(lián)合是否通過(guò)抑制內(nèi)質(zhì)網(wǎng)應(yīng)激特有分子伴侶進(jìn)而探討其可抑制內(nèi)質(zhì)網(wǎng)應(yīng)激誘導(dǎo)的細(xì)胞凋亡從而發(fā)揮其對(duì)CIAKI后的腎功能的保護(hù)作用,并從藥物聯(lián)合角度進(jìn)一步間接明確叢集素在CIAKI發(fā)生中的診斷意義及不同藥物聯(lián)合對(duì)其干預(yù)作用。研究方法:Wistar大鼠50只,隨機(jī)分成5組:A組(阿托伐他汀組,n=10);P組(普羅布考+阿托伐他汀組,n=10);Q組(前列地爾+阿托伐他汀組,n=10);NC組(對(duì)比劑組,n=10);N組(對(duì)照組,n=10)。常溫下適應(yīng)性喂養(yǎng)7天后,1周末鎖骨下靜脈取血2ml測(cè)腎功能。予P組普羅布考灌胃14天后與阿托伐他汀同時(shí)灌胃10天。Q組與A組等體積0.5%羧甲基纖維素鈉灌胃14天后,改用阿托伐他汀灌胃10天,N組與NC組每天等體積0.5%羧甲基纖維素鈉溶液灌胃。灌胃21天后,第22天開始自鎖骨下靜脈注射泛影葡胺,N組注射生理鹽水;Q組分別于注射對(duì)比劑前24小時(shí)、術(shù)前30分鐘、術(shù)后24小時(shí)鎖骨下靜脈注射前列地爾,其他組等劑量生理鹽水鎖骨下靜脈注射,術(shù)后繼續(xù)灌胃直至處死。分別于注射對(duì)比劑后48小時(shí)及72小時(shí)取血2ml測(cè)腎功能,72小時(shí)后處死大鼠,取出腎臟,左腎用福爾馬林固定,石蠟包埋送往病理科。右腎置于-80度液氮冰箱冷凍保存用于western blot及real-time RT-PCR實(shí)驗(yàn)。腎臟病理切片HE染色法及電鏡下檢測(cè)腎臟病理變化,用TUNEL染色法檢測(cè)腎臟細(xì)胞凋亡。western blot及免疫組化檢測(cè)內(nèi)質(zhì)網(wǎng)應(yīng)激靶點(diǎn)GRP78、Clusterin、GADD153/CHOP、Caspase-12在蛋白水平上的表達(dá),real-time RT-PCR檢測(cè)上述靶點(diǎn)在核酸水平的表達(dá)。統(tǒng)計(jì)分析應(yīng)用SPSS19.0統(tǒng)計(jì)軟件。以均值±標(biāo)準(zhǔn)差(x±s)衡量計(jì)量資料數(shù)值,配對(duì)樣本t檢驗(yàn)方法比較組內(nèi)數(shù)據(jù);單因素方差分析法、Mann-Whitney U檢驗(yàn)或Kruksal-Wallis檢驗(yàn)比較組間數(shù)據(jù)。P㩳0.05表示差異有統(tǒng)計(jì)學(xué)意義。研究結(jié)果:1、泛影葡胺能引起大鼠腎功能肌酐指標(biāo)明顯增高,NC組造模前后差異值最大。阿托伐他汀組及聯(lián)合普羅布考或前列地爾組肌酐升高值較對(duì)比劑組明顯降低,聯(lián)合用藥組與空白組無(wú)明顯差異,其中Q組對(duì)腎保護(hù)作用明顯好于阿托伐他汀單藥組。2、NC組GRP78、GADD153/CHOP及Caspase-12核酸表達(dá)水平最高,其中在GADD153/CHOP及Caspase-12的表達(dá)與其余四組相比有明顯統(tǒng)計(jì)學(xué)差異;而對(duì)于聯(lián)合用藥及單藥組之間的比較無(wú)明顯統(tǒng)計(jì)學(xué)差異。3、Western blot實(shí)驗(yàn)中NC組GRP78、GADD153及Caspase-12蛋白表達(dá)量較其他四組顯著升高,A組在GRP78靶點(diǎn)蛋白表達(dá)量明顯高于P組,在GADD153明顯高于聯(lián)合用藥組,在Caspase-12高于Q組。在GADD153和Caspase-12的表達(dá)Q組蛋白表達(dá)量明顯低于P組,聯(lián)合用藥組在以上靶點(diǎn)的表達(dá)接近于N組甚至低于N組。4、在病理HE染色及電鏡中,NC組中腎小管上皮細(xì)胞空泡樣改變,部分上皮細(xì)胞壞死、基底膜增厚,腎小管腔狹窄,內(nèi)質(zhì)網(wǎng)及線粒體數(shù)量減少、發(fā)生腫脹,管腔面微絨毛雜亂脫落;而阿托伐他汀組及聯(lián)合用藥組腎小管損傷程度較對(duì)比劑組減輕,聯(lián)合用藥組病例損傷程度低于阿托伐他汀組。5、凋亡指數(shù):NC組A組Q組P組N組,差異有統(tǒng)計(jì)學(xué)意義,其中A組較聯(lián)合用藥組顯著升高。P組與Q組無(wú)明顯差異。6、免疫組化:NC組GRP78、GADD153、Caspase-12表達(dá)量最高,A組在GRP78的表達(dá)上略高于聯(lián)合用藥組,無(wú)明顯統(tǒng)計(jì)學(xué)差異,在GADD153的表達(dá)上高于聯(lián)合用藥組,與Q組有明顯差異,在Caspase-12表達(dá)上與P組和Q組有明顯差異。7、叢集素核酸表達(dá)五組間未見明顯差異。NC組在蛋白表達(dá)水平明顯高于其他四組,A組也顯著高于聯(lián)合用藥組,Q組低于P組的表達(dá)。研究結(jié)論:1、內(nèi)質(zhì)網(wǎng)應(yīng)激誘導(dǎo)的細(xì)胞凋亡通路在泛影葡胺誘導(dǎo)的急性腎損傷的發(fā)病中發(fā)揮了作用;阿托伐他汀、普羅布考及前列地爾對(duì)大鼠CIAKI的發(fā)病起到預(yù)防作用,這種作用可能是通過(guò)抑制內(nèi)質(zhì)網(wǎng)應(yīng)激誘導(dǎo)的細(xì)胞凋亡通路而實(shí)現(xiàn)的,其中普羅布考、前列地爾與阿托伐他汀聯(lián)合對(duì)對(duì)比劑腎損傷的腎保護(hù)作用可能優(yōu)于阿托伐他汀組,前列地爾與阿托伐他汀的效果更明顯。2、進(jìn)一步闡明了,叢生蛋白是可以早期預(yù)測(cè)泛影葡胺誘導(dǎo)的急性腎損傷發(fā)生的新型生物標(biāo)志物,普羅布考及前列地爾與阿托伐他汀的聯(lián)合作用使CIAKI中叢生蛋白的表達(dá)下調(diào)證明其對(duì)泛影葡胺誘導(dǎo)的急性腎損傷后腎保護(hù)作用強(qiáng)于阿托伐他汀,其中前列地爾聯(lián)合阿托伐他汀效果更顯著。
[Abstract]:Research background and purpose: contrast-induced acute kidney injury (CIAKI) induced by contrast agent, which is also known as contrast agent nephropathy (contrast induced nephropathy, CIN), is an important clinical complication after using contrast agent. It is followed by percutaneous coronary intervention after stent thrombosis and stent restenosis. The third major postoperative complications. Clinical use of contrast agents mainly including meglumine, iodifol, iodiprol and other.CIAKI mechanisms are very complicated. Studies have shown that endoplasmic reticulum stress and induced apoptosis play an important role in CIAKI renal tubule injury. Antioxidation and anti-inflammatory or improved renal vascular perfusion can inhibit the apoptosis of renal tubular cells and may prevent the occurrence of CIAKI. Previous studies have shown that atorvastatin could successfully inhibit the apoptosis induced by endoplasmic reticulum stress and prevent the occurrence of CIAKI. However, whether the combined use of the combined drug can inhibit endoplasmic reticulum stress induced stress. There is no specific study on the role of apoptosis to prevent the occurrence of CIAKI. In recent decades, many early new markers to detect CIAKI have been found. Previous studies have preliminarily demonstrated that the cluster protein can predict the occurrence of CIAKI, but lack of further demonstration. Therefore, this study is to establish a rat CIAKI model by using meglumine. The effects of drug combination and atorvastatin on the renal injury of rat contrast agent were compared, and the effects of various drugs on the inhibition of endoplasmic reticulum stress specific molecular chaperone and the inhibition of the apoptosis induced by endoplasmic reticulum stress were explored to protect the renal function after CIAKI. The combined angle of drugs was used to further clarify the diagnostic significance of cluster in the occurrence of CIAKI and the effect of the combination of different drugs. Research methods: 50 Wistar rats were randomly divided into 5 groups: A group (atorvastatin group, n=10); P group (probucol + atorvastatin group, n= 10); Q group (alprostadil + atorvastatin group, n=10); group NC (contrast agent) Group, n=10, group N (control group, n=10). After 7 days of adaptive feeding at normal temperature, the function of 2ml was measured by the subclavian vein for the 1 weekend. In group P, 14 days after filling the stomach with atorvastatin for 10 days at the same time, group.Q and A group 0.5% carboxymethyl cellulose sodium for 14 days, with atorvastatin for 10 days, N group and NC group per day 0.5. 21 days after gavage, the subclavicular vein was injected into the subosseous vein for 21 days and the saline was injected into the subosseous vein on the twenty-second day. Group Q was injected with the contrast agent 24 hours before the injection, 30 minutes before the operation, 24 hours after the operation, and the other groups were injected with the subclavian vein, and the other groups were injected with the subclavian vein, and the gastric perfusion continued after the operation. After 48 hours and 72 hours after injection of contrast agent, the renal function was measured by 2ml. After 72 hours, rats were killed and kidney was taken out. The left kidney was fixed with formalin and paraffin was buried in the disease science. The right kidney was stored in -80 degree liquid nitrogen refrigerator for cryopreservation for western blot and real-time RT-PCR experiment. Renal pathological section HE staining and electron microscopy To detect renal pathological changes, TUNEL staining was used to detect.Western blot of renal cell apoptosis and the expression of GRP78, Clusterin, GADD153/CHOP, Caspase-12 at protein level in endoplasmic reticulum stress target, and real-time RT-PCR was used to detect the expression of the target at the level of nucleic acid. Statistical analysis applied SPSS19.0 software. Standard deviation (x + s) measure measurement data, paired sample t test method to compare intra group data; single factor variance analysis, Mann-Whitney U test or Kruksal-Wallis test comparison group data.P? 0.05 showed significant difference. 1, the results of the study were that the index of renal functional creatinine increased significantly in rats, and the model of NC group was created. The creatinine elevation value of atorvastatin group and combined proponol group or prostadil group was significantly lower than that of the contrast group. There was no significant difference between the combination group and the blank group. The protective effect of the Q group was better than that of the Atorvastatin single drug group.2, and the GRP78, GADD153/CHOP and Caspase-12 nucleic acids in the group NC were the highest. There was significant difference in the expression of GADD153/CHOP and Caspase-12 compared with the other four groups, but there was no significant difference between the combination and the single drug group.3. The expression of GRP78, GADD153 and Caspase-12 protein in group NC was significantly higher than that of the other four groups in the Western blot experiment, and the expression of the A group in GRP78 target protein was significantly higher than that of P. The expression of Q histone expression in GADD153 and Caspase-12 was significantly lower than that of group P in the group of GADD153. The expression of histone in GADD153 and Caspase-12 was significantly lower than that in group P. The expression of the combined drug group at the above target was close to that of the N group and even lower than the N group.4. In the pathological HE staining and the electrical mirror, the vacuoles like changes in the renal tubular epithelial cells in the NC group and some epithelial cells in the NC group. Necrosis, thickening of basal membrane, narrowing of renal tubule, decreased number of endoplasmic reticulum and mitochondria, swelling and tumbling of microvilli in the lumen, and reduction of renal tubule injury in atorvastatin group and combined drug group compared with contrast agent group, the degree of injury in the combined drug group was lower than that of atorvastatin group.5, apoptosis index: group Q P group N of group A group of group NC, group N, There was significant difference in the difference between group A and group Q. There was no significant difference between group.P and group Q,.6, immunohistochemistry: the expression of GRP78, GADD153, Caspase-12 in NC group was the highest, and the expression of A group in GRP78 was slightly higher than that of the combined drug group, and there was no significant difference in the expression of GADD153. The expression of spase-12 was significantly different from that of the P group and the Q group. There was no significant difference between the five groups. The expression level of the group.NC was significantly higher than that of the other four groups, and the A group was also significantly higher than the combination group, and the Q group was lower than the P group. 1, the apoptosis pathway induced by endoplasmic reticulum stress was in the acute kidney induced by meglumine. It plays a role in the pathogenesis of injury; atorvastatin, probucol and alprostadil play a preventive role in the pathogenesis of CIAKI in rats. This effect may be achieved by inhibiting the apoptosis pathway induced by endoplasmic reticulum stress, including propriopol, alprostadil and atorvastatin in the renal protection of contrastive renal injury. The effect of alprostadil and atorvastatin is more obvious than the Atorvastatin group, and the effect of alprostadil and atorvastatin is more obvious.2, further clarifying that the cluster protein is a new biomarker for the early prediction of acute renal injury induced by meglumine. The combination of probucol and alprostadil and atorvastatin makes the expression of CIAKI clump protein. The protective effect of alprostadil combined with atorvastatin was stronger than that of atorvastatin after kidney injury induced by meglumine dihydrochloride.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R692

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