P38MAPK通路在草酸钙肾结石形成中的机制研究

发布时间：2018-06-09 17:16

本文选题：草酸钙肾结石 + 草酸　；参考：《天津医科大学》2017年硕士论文

【摘要】：目的探讨激活后的NLRP3炎症小体进一步促进草酸钙肾结石形成的机制。方法通过对正常肾组织与草酸钙结石患者肾组织进行免疫组化检测P38蛋白表达水平差异;培养NRK-52E细胞,采用不同浓度的P38MAPK通路抑制剂(SB203580)(0,20,40,60,80,1.00umol/L)和不同浓度的草酸(0,0.2,0.4,0.6,0.8,1.0,mmol/L)分别作用于NRK-52E细胞24h后,分别采用检测培养基中LDH的含量的方法和MTT法检测草酸和SB203580对NRK-52E细胞的毒性;草酸以及添加SB203580的草酸处理NRK-52E细胞24h后,使用免疫荧光共聚焦显微镜观察细胞P38蛋白表达水平差异;草酸以及添加SB203580的草酸处理NRK-52E细胞24h后,相差显微镜下观察NRK-52E细胞表面晶体粘附性变化,并采用Western blotting和RT-q PCR法分析对NLRP3、P38及黏附因子OPN蛋白水平及m RNA表达量的影响;草酸处理NRK-52E细胞24h后,使用线粒体绿色荧光探针检测NRK-52E细胞内线粒体变化情况;通过转染NLRP3-Si RNA将NLRP3基因沉默之后,再次给予草酸处理24h后,采用western blotting实验方法检测NLRP3、P38以及OPN蛋白的表达水平差异,采用RT-q PCR法分别检测CD44、OPN、HAS1、HAS2以及HAS3m RNA表达水平变化情况;通过直接给予SD大鼠饮用乙二醇水从而构建草酸钙肾结石模型,采用透视电镜观察大鼠肾小管上皮细胞内细胞器变化情况,并采用Western blotting法检测大鼠肾组织中NLRP3、P38及OPN蛋白表达差异,通过免疫组化检测结石组大鼠肾组织和对照组大鼠肾组织P38蛋白表达水平差异,采用RT-q PCR法检测NLRP3、P38、OPN、HAS1、HAS2、HAS3、以及CD44 m RNA表达水平变化。所有实验数据采用SPSS 20.0软件进行统计分析。结果免疫组化显示P38蛋白在草酸钙肾结石患者肾组织中的表达水平明显高于正常肾组织标本,进而组织水平说明了P38MAPK通路的参与草酸钙肾结石形成过程;测定细胞培养基中LDH含量结果显示,当草酸浓度为0.8mmol/L处理NRK-52E细胞24h后,培养基内LDH的含量明显高于草酸浓度在0-0.6mmol/L(P0.05),当SB203580浓度为60umol/L处理NRK-52E细胞24h后,培养基内LDH的含量明显高于SB203580浓度在0-40umol/L(P0.05),同样MTT法结果显示SB203580浓度为60umol/L时(P0.05)、草酸浓度为0.8mmol/L时(P0.05),对肾小管上皮细胞具有明显的细胞毒性;草酸处理NRK-52E细胞24h后,免疫荧光共聚焦显微镜下显示草酸组细胞P38蛋白表达水平明显多于对照组;草酸以及添加SB203580的草酸处理NRK-52E细胞24h后,western blotting结果发现草酸组细胞中NLRP3、P38以及黏附因子OPN的蛋白质表达水平比完全对照组增高(P0.05),而草酸+抑制剂组与完全对照组相比仅有NLRP3蛋白表达水平增高(P0.05),P38和黏附因子OPN蛋白未见明显升高(P0.05);草酸(0.6mmol/L)处理NRK-52E细胞24h后,使用线粒体绿色荧光探针检测发现NRK-52E细胞内线粒体明显肿胀;通过NLRP3-Si RNA转染NRK-52E细胞后,草酸处理24小时后,NLRP3蛋白、P38蛋白以及粘附因子OPN蛋白表达水平下降(P0.05),细胞表面的黏附因子CD44、HAS1、HAS2、HAS3、OPN的表达水平显著降低(P0.05);通过乙二醇饮水法构建SD大鼠草酸钙肾结石模型,利用透视电镜观察发现高草酸尿症大鼠模型肾小管上皮细胞内细胞核成椭圆形,核内易染色质凝聚边集,线粒体肿胀空泡化,免疫组化结果发现P38蛋白在高草酸尿症大鼠肾组织中表达水平均较对照组肾组织显著增高,western boltting检测发现结石组大鼠的肾组织标本中NLRP3、P38、OPN蛋白表达水平显著高于正常大鼠肾组织(P0.05),RT-q PCR技术发现高草酸尿症大鼠的肾组织标本中P38、NLRP3、Caspase-1、IL-1β、HAS1、HAS2、HAS3、CD44、OPN的m RNA表达量也显著高于正常大鼠肾组织(P0.05)。结论草酸能够引起肾小管上皮细胞发生“细胞焦亡”;P38MAPK通路参与草酸钙肾结石的形成;激活的NLRP3炎症小体是通过P38MAPK通路影响肾小管上皮细胞表面黏附因子的改变,进而促进肾结石的形成,从而为草酸钙结石的治疗和预防提供了理论依据。
[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.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R692.4

【参考文献】