本文選題：草酸鈣腎結(jié)石 + 草酸��； 參考：《天津醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的探討激活后的NLRP3炎癥小體進(jìn)一步促進(jìn)草酸鈣腎結(jié)石形成的機(jī)制。方法通過(guò)對(duì)正常腎組織與草酸鈣結(jié)石患者腎組織進(jìn)行免疫組化檢測(cè)P38蛋白表達(dá)水平差異;培養(yǎng)NRK-52E細(xì)胞,采用不同濃度的P38MAPK通路抑制劑(SB203580)(0,20,40,60,80,1.00umol/L)和不同濃度的草酸(0,0.2,0.4,0.6,0.8,1.0,mmol/L)分別作用于NRK-52E細(xì)胞24h后,分別采用檢測(cè)培養(yǎng)基中LDH的含量的方法和MTT法檢測(cè)草酸和SB203580對(duì)NRK-52E細(xì)胞的毒性;草酸以及添加SB203580的草酸處理NRK-52E細(xì)胞24h后,使用免疫熒光共聚焦顯微鏡觀察細(xì)胞P38蛋白表達(dá)水平差異;草酸以及添加SB203580的草酸處理NRK-52E細(xì)胞24h后,相差顯微鏡下觀察NRK-52E細(xì)胞表面晶體粘附性變化,并采用Western blotting和RT-q PCR法分析對(duì)NLRP3、P38及黏附因子OPN蛋白水平及m RNA表達(dá)量的影響;草酸處理NRK-52E細(xì)胞24h后,使用線(xiàn)粒體綠色熒光探針檢測(cè)NRK-52E細(xì)胞內(nèi)線(xiàn)粒體變化情況;通過(guò)轉(zhuǎn)染NLRP3-Si RNA將NLRP3基因沉默之后,再次給予草酸處理24h后,采用western blotting實(shí)驗(yàn)方法檢測(cè)NLRP3、P38以及OPN蛋白的表達(dá)水平差異,采用RT-q PCR法分別檢測(cè)CD44、OPN、HAS1、HAS2以及HAS3m RNA表達(dá)水平變化情況;通過(guò)直接給予SD大鼠飲用乙二醇水從而構(gòu)建草酸鈣腎結(jié)石模型,采用透視電鏡觀察大鼠腎小管上皮細(xì)胞內(nèi)細(xì)胞器變化情況,并采用Western blotting法檢測(cè)大鼠腎組織中NLRP3、P38及OPN蛋白表達(dá)差異,通過(guò)免疫組化檢測(cè)結(jié)石組大鼠腎組織和對(duì)照組大鼠腎組織P38蛋白表達(dá)水平差異,采用RT-q PCR法檢測(cè)NLRP3、P38、OPN、HAS1、HAS2、HAS3、以及CD44 m RNA表達(dá)水平變化。所有實(shí)驗(yàn)數(shù)據(jù)采用SPSS 20.0軟件進(jìn)行統(tǒng)計(jì)分析。結(jié)果免疫組化顯示P38蛋白在草酸鈣腎結(jié)石患者腎組織中的表達(dá)水平明顯高于正常腎組織標(biāo)本,進(jìn)而組織水平說(shuō)明了P38MAPK通路的參與草酸鈣腎結(jié)石形成過(guò)程;測(cè)定細(xì)胞培養(yǎng)基中LDH含量結(jié)果顯示,當(dāng)草酸濃度為0.8mmol/L處理NRK-52E細(xì)胞24h后,培養(yǎng)基內(nèi)LDH的含量明顯高于草酸濃度在0-0.6mmol/L(P0.05),當(dāng)SB203580濃度為60umol/L處理NRK-52E細(xì)胞24h后,培養(yǎng)基內(nèi)LDH的含量明顯高于SB203580濃度在0-40umol/L(P0.05),同樣MTT法結(jié)果顯示SB203580濃度為60umol/L時(shí)(P0.05)、草酸濃度為0.8mmol/L時(shí)(P0.05),對(duì)腎小管上皮細(xì)胞具有明顯的細(xì)胞毒性;草酸處理NRK-52E細(xì)胞24h后,免疫熒光共聚焦顯微鏡下顯示草酸組細(xì)胞P38蛋白表達(dá)水平明顯多于對(duì)照組;草酸以及添加SB203580的草酸處理NRK-52E細(xì)胞24h后,western blotting結(jié)果發(fā)現(xiàn)草酸組細(xì)胞中NLRP3、P38以及黏附因子OPN的蛋白質(zhì)表達(dá)水平比完全對(duì)照組增高(P0.05),而草酸+抑制劑組與完全對(duì)照組相比僅有NLRP3蛋白表達(dá)水平增高(P0.05),P38和黏附因子OPN蛋白未見(jiàn)明顯升高(P0.05);草酸(0.6mmol/L)處理NRK-52E細(xì)胞24h后,使用線(xiàn)粒體綠色熒光探針檢測(cè)發(fā)現(xiàn)NRK-52E細(xì)胞內(nèi)線(xiàn)粒體明顯腫脹;通過(guò)NLRP3-Si RNA轉(zhuǎn)染NRK-52E細(xì)胞后,草酸處理24小時(shí)后,NLRP3蛋白、P38蛋白以及粘附因子OPN蛋白表達(dá)水平下降(P0.05),細(xì)胞表面的黏附因子CD44、HAS1、HAS2、HAS3、OPN的表達(dá)水平顯著降低(P0.05);通過(guò)乙二醇飲水法構(gòu)建SD大鼠草酸鈣腎結(jié)石模型,利用透視電鏡觀察發(fā)現(xiàn)高草酸尿癥大鼠模型腎小管上皮細(xì)胞內(nèi)細(xì)胞核成橢圓形,核內(nèi)易染色質(zhì)凝聚邊集,線(xiàn)粒體腫脹空泡化,免疫組化結(jié)果發(fā)現(xiàn)P38蛋白在高草酸尿癥大鼠腎組織中表達(dá)水平均較對(duì)照組腎組織顯著增高,western boltting檢測(cè)發(fā)現(xiàn)結(jié)石組大鼠的腎組織標(biāo)本中NLRP3、P38、OPN蛋白表達(dá)水平顯著高于正常大鼠腎組織(P0.05),RT-q PCR技術(shù)發(fā)現(xiàn)高草酸尿癥大鼠的腎組織標(biāo)本中P38、NLRP3、Caspase-1、IL-1β、HAS1、HAS2、HAS3、CD44、OPN的m RNA表達(dá)量也顯著高于正常大鼠腎組織(P0.05)。結(jié)論草酸能夠引起腎小管上皮細(xì)胞發(fā)生“細(xì)胞焦亡”;P38MAPK通路參與草酸鈣腎結(jié)石的形成;激活的NLRP3炎癥小體是通過(guò)P38MAPK通路影響腎小管上皮細(xì)胞表面黏附因子的改變,進(jìn)而促進(jìn)腎結(jié)石的形成,從而為草酸鈣結(jié)石的治療和預(yù)防提供了理論依據(jù)。
[Abstract]:Objective to explore the mechanism of further promoting the formation of calcium oxalate nephrolithiasis by activated NLRP3 inflammatory bodies. Methods the difference in the expression of P38 protein was detected by immunohistochemistry in renal tissue of normal renal tissue and calcium oxalate stone patients, and NRK-52E cells were cultured, and P38MAPK pathway inhibitor (SB203580) was used at different concentrations (0,20,40,60,80,1.00um). Ol/L) and different concentrations of oxalic acid (0,0.2,0.4,0.6,0.8,1.0, mmol/L) acted on 24h of NRK-52E cells, respectively, to detect the toxicity of oxalic acid and SB203580 to NRK-52E cells by the method of detecting the content of LDH in the medium and MTT method respectively. After the oxalic acid and the oxalic acid added SB203580 in the NRK-52E cell 24h, the immunofluorescence confocal microscopy was used. The difference of P38 protein expression level was observed by microscope; oxalic acid and oxalic acid with SB203580 were used to treat NRK-52E cell 24h, and the changes of crystal adhesion on the surface of NRK-52E cells were observed by phase contrast microscope, and the effect of Western blotting and RT-q PCR method on NLRP3, P38 and adhesion factor OPN egg white level and expression quantity were analyzed. Oxalic acid treatment After 52E cell 24h, the mitochondrial changes in NRK-52E cells were detected by the mitochondrial green fluorescence probe. After transfection of NLRP3-Si RNA, the NLRP3 gene was silenced and 24h was treated again by oxalic acid. Western blotting test method was used to detect NLRP3, P38 and OPN protein levels. The expression level of HAS1, HAS2 and HAS3m RNA was changed. The calcium oxalate nephrolithiasis model was constructed by drinking glycol water from SD rats directly. The changes of organelles in the renal tubular epithelial cells of rats were observed by fluoroscopy and the Western blotting method was used to detect the difference in the expression of NLRP3, P38 and OPN proteins in the renal tissue of rats. The expression level of P38 protein in kidney tissue and control group of rats was detected by immunohistochemistry. The changes of NLRP3, P38, OPN, HAS1, HAS2, HAS3, and CD44 m RNA were detected by RT-q PCR method. All the experimental data were analyzed by 20 software. The level of expression in renal tissue was significantly higher than that of normal renal tissue, and the level of P38MAPK pathway involved in the formation of calcium oxalate nephrolithiasis, and the results of LDH content in the cell culture medium showed that the content of LDH in cultured NRK-52E cells was significantly higher than the concentration of oxalic acid in 0-0.6 after the concentration of oxalic acid was 0.8mmol/L treatment 24h. Mmol/L (P0.05), when the concentration of SB203580 was 60umol/L treated NRK-52E cell 24h, the content of the cultured LDH was significantly higher than that of SB203580 concentration at 0-40umol/L (P0.05). The same MTT method showed that the concentration of SB203580 was at the time of the concentration of oxalic acid. After RK-52E cell 24h, the expression of P38 protein in oxalic acid group was significantly higher than that of the control group. After oxalic acid and oxalic acid added SB203580, Western blotting found NLRP3 in oxalic acid group, P38 and OPN protein expression level of adhesion promoter was higher than that in complete control group. High (P0.05), and oxalic acid + inhibitor group only increased the expression level of NLRP3 protein (P0.05), P38 and adhesion factor OPN protein did not significantly increase (P0.05). Oxalic acid (0.6mmol/L) after NRK-52E cell 24h, the mitochondrial fluorescence probe detection found that the mitochondria in NRK-52E cells obviously swelling; through NLRP3-Si RNA. After transfection of NRK-52E cells, after 24 hours of oxalic acid treatment, the expression level of NLRP3 protein, P38 protein and adhesion factor OPN protein decreased (P0.05). The expression level of adhesion factor CD44, HAS1, HAS2, HAS3, OPN of cell surface decreased significantly (P0.05). The calcium oxalate kidney stone model of SD rats was constructed by drinking water method of ethylene glycol, and the findings were observed by perspective electron microscopy. The nucleus of renal tubular epithelial cells in the rat model of high oxalinuria was elliptical, the nucleus of chromatin condensed in the nucleus and the vacuolization of mitochondria. The immunohistochemical results showed that the expression level of P38 protein in the renal tissue of the rats with high oxalinuria was significantly higher than that of the control group. Western boltting detection found the kidney of the rats in the stone group. The expression level of NLRP3, P38, OPN protein was significantly higher than that of normal rat kidney tissue (P0.05). RT-q PCR technology found that the expression of P38, NLRP3, Caspase-1, IL-1 beta, HAS1, HAS1, HAS1, HAS1, HAS1, HAS2, and PCR in the kidney tissue of the rats with high oxalic urine were also significantly higher than that of normal rats. The P38MAPK pathway involved in the formation of calcium oxalate nephrolithiasis, and the activated NLRP3 inflammatory corpuscle affects the change of the surface adhesion factor of the renal tubular epithelial cells through the P38MAPK pathway, thus promoting the formation of renal calculi, thus providing a theoretical basis for the treatment and prevention of calcium oxalate stones.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R692.4

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本文編號(hào)：2000510