【作者】 王博涵；

【导师】 叶章群； 陈志强； 余虓；

【作者基本信息】 华中科技大学 ， 外科学， 2013， 博士

【摘要】 目的：探讨草酸和一水草酸钙对人肾小管上皮细胞HK-2细胞的毒性作用。方法：共培养体系培养HK-2细胞至长满后,使用不同浓度（0,1,5和10mmmmol/L）的草酸和一水草酸钙处理HK-2细胞24h,通过检测细胞培养基中的乳酸脱氢酶LDH的含量和DAPI染色结果分析草酸及一水草酸钙对细胞的毒性作用。结果：DAPI染色显示草酸浓度在0-5mmol/L时,细胞数目没明显变化,5mmol/L时个别细胞的细胞核呈固缩状,草酸浓度10mmol/L时,细胞数目开始明显减少。水草酸钙浓度在1mmol/L时细胞数目就开始明显减少,5mmol/L和10mmo1/L时细胞基本死亡。5mmol/L和10mmol/L草酸在处理HK-2细胞24h后培养基中的LDH含量显著高于未处理组（p<0.01）。1mmo1/L、5mmol/L和10mmol/L一水草酸钙组培养基中LDH含量均显著高于未处理组（p<0.01）。结论：草酸和一水草酸钙对HK-2细胞的毒性呈浓度相关性。一水草酸钙晶体相比较草酸更具有细胞毒性。目的：探讨一水草酸钙晶体（calcium oxalate monohydrate, COM）通过对人肾小管上皮细胞HK-2的作用能否激活MAPK信号通路,通过拮抗MAPK信号通路能否减少透明质酸合成酶、CD44的产生以及能否抑制COM晶体对细胞的粘附作用。方法：HK-2细胞在饥饿处理12h后分别在不同时间点（0min、15mmin、30mmin、1h和2h）暴露在COM晶体（1mmol/L）及阳性对照下。检测p38MAPK.JNK和ERK蛋白的磷酸化情况。p38MAPK通路的抑制剂SB203580对细胞进行干预,通过western blot和RT-PCR技术检测p38-MAPK磷酸化情况及透明质酸合成酶1-3和CD44的mRNA表达情况。通过离子色谱仪检测COM晶体与肾小管上皮细胞的粘附情况。结果：HK-2细胞在饥饿处理12h后,COM晶体（1mmol/L）能快速的、充分的激活p38MAPK信号通路。COM晶体能轻度激活JNK信号途径但是无法激活ERK信号途径。通过使用p38MAPK信号途径的抑制剂SB203580（50μM）能够充分阻滞p38MAPK信号通路的激活并且能够降低透明质酸合成酶1-3和CD44的mRNA表达以及减少肾小管上皮细胞与COM晶体的粘附。结论：一水草酸钙晶体能够选择性激活p38MAPK和JNK信号途径。p38MAPK信号通路抑制剂SB203580能够降低透明质酸合成酶1-3和CD44的mRNA表达以及减少肾小管上皮细胞与COM晶体的粘附。目的：观察透明质酸（HA）、CD44及透明质酸合成酶（HAS1-3）在结石患者肾组织中的表达,着重探讨HAS1在一水草酸钙晶体（calcium oxalate monohydrate, COM）和人肾小管上皮细胞在粘附过程中的作用。方法：通过免疫组化的方法,检测结石患者肾组织和正常肾组织中CD44、HAS1-3和HA的表达差异。通过RT-PCR和western blot检测I-IK-2细胞在分别在不同时间点（Oh、24h、48h）暴露在COM晶体（lmmol/L）下HAS1的mRNA和蛋白表达情况。通过ELISA方法,检测HK-2细胞在不同时间点（0h、12h、24h、36h、48h）暴露于COM晶体后培养基中HA的含量。通过干扰HAS1,检测其对HA的合成以及对肾小管上皮与COM晶体粘附力的影响。通过共聚焦显微镜和离子色谱仪检测COM晶体与肾小管上皮细胞的粘附情况。结果：免疫组化结果显示在结石患者肾组织中,CD44、HAS1-3及HA比正常肾组织都有明显升高。HK-2细胞在COM晶体（lmmol/L）处理24h后,HAS1-3和CD44的mRNA表达最高,48h后下降,其中HAS1的表达升高最明显。HK-2细胞在COM晶体（1mmol/L）处理24h后HAS1的蛋白含量开始升高,48h达到最高。通过对HAS1的siRNA干扰能充分的降低HAS1的表达。HK-2细胞在COM晶体（1mmol/L）处理24h后组织培养基中的HA的含量达到最高,其后开始下降。荧光共聚焦染色和离子色谱仪结果显示,通过抑制HAS1的表达可以大大降低肾小管上皮细胞与COM晶体的粘附。结论：结石患者肾组织中CD44、HAS1-3和HA都有高表达。透明质酸在COM晶体与肾小管上皮的粘附过程中起到非常重要的作用,通过抑制HAS1的表达可以减少HA的表达从而降低肾小管上皮细胞对COM晶体的粘附。目的：长链非编码RNAs （lncRNAs）具有非常重要的生物功能。本研究通过lncRNA芯片研究一水草酸钙刺激人近端肾小管后lncRNAs的变化,为将来结石形成机制的研究提供候选lncRNAs。方法：通过lncRNA芯片检测lmmo1/L一水草酸钙刺激人近端肾小管HK-2细胞24h后长链非编码RNA的变化情况。通过qPCR的方法验证芯片结果。结果：经过1mmol/L一水草酸钙刺激人近端肾小管HK-2细胞24h后2971条lncRNAs发生了变化,其中上调1630条,下调1341条。通过qPCR的方法验证了4条lncRNAs （ENST00000430583、ENST00000428930、NR₀29401和chr13）。结果显示qPCR结果和基因芯片结果具有良好的一致性。结论：本研究首次报道肾小管上皮细胞经一水草酸钙刺激24h后lncRNAs的变化,结果中表达有差异的lncRNAs可能在结石形成过程中有巨大的作用。综上所述,本研究为结石形成的病理过程提供新的思路,为将来泌尿系结石形成的机制研究提供理论依据。更多还原

【Abstract】 Objective To evaluate the toxic effect of oxalate and calcium oxalate monohydrate （COM） crystals on human renal tubular epithelial cells （HK-2）.Methods HK-2cells were cultured in co-culture system to confluence. Oxalic acid and COM crystals with different concentration （0,1,5and10mmol/L） were then added. The toxic effect of Oxalic acid and COM crystals on HK-2cells at24h after incubation were examined by measuring the activity of lactic dehydrogenase （LDH） and dyeing with4’,6-diamidino-2-phenylindole（DAPI）.Results Dyeing with DAPI showed that the number of cells was not significant decreased until the concentration of oxalic acid up to10mmol/L. The number of cells was reduced dramatically after treating with1mmol/L COM. Cells were almost dead after incubation24h with5and10mmol/L COM. LDH activity was increased significantly in5and10mmol/L oxalic acid group （p<0.01）. Comparing with oxalic acid group, LDH activity was increased significantly in1,5and10mmol/L COM group （p<0.01）.Conclusion Oxalic acid and COM crystals have toxic effect on HK-2cells in a concentration dependent manner. COM crystals are more toxic than oxalic acid. Objective To evaluate the MAPK signaling pathways involved in the process of calcium oxalate monohydrate （COM） crystals in human renal tubular epithelial cells （HK-2）. To research the effect of SB203580on regulation of HAS1-3, CD44and adhesive influence between COM crystal and HK-2cells.Methods HK-2cells were cultured in serum-starved DMEM for12h before exposure to calcium oxalate monohydrate （lmM） for different time course （0,15min,30min,60min and120min）. The activity of MAPK signaling pathways were evaluated by phosphorylation of p38, JNK and ERK. SB203580was used to block the transduction of p38. RT-PCR was used to measure the mRNA level of HAS1-3and CD44. Ion chromatograph was performed to measure the adhesion effect between COM crystal and HK-2cells.Results Exposure to calcium oxalate monohydrate （COM） crystal rapidly activated p38-MAPK. Calcium oxalate monohydrate （COM） crystal induced modest activation of JNK. In contrast, COM crystal had no effect on phosphorylation of ERK. The adhesive effect between COM crystal and HK-2cells was reduced by the use of SB203580（50μM）.The mRNA levels of HAS1-3and CD44were diminished by the use of SB203580. Conclusion Exposure to calcium oxalate monohydrate （COM） crystal selectively activates p38-MAPK and JNK signaling pathways. SB203580reduces the mRNA levels of HAS1-3, CD44and diminish the adhesive effect between COM crystal and HK-2cells. Objective To evaluate the expression of hyaluronan, CD44and hyaluronan synthase1-3in kidney stone patients. The effect of HAS1on the adhesion between renal epithelial cells （HK-2） and calcium oxalate monohydrate （COM） crystals was studied. Methods The expression of hyaluronan, CD44and hyaluronan synthase1-3were measured between kidney stone patients and normail kidney patients by immunohistochemistry. HK-2cells were exposed to calcium oxalate monohydrate （1mM） for different time course （Oh,24h,48h）. The mRNA levels of HAS1-3and CD44were measured by RT-PCR, and the protein level of HAS1was measured by western blot. The expression of hyaluronan was detected by Elisa kit. siRNA methods was used to influent the content of hyaluronan and the adhesion of renal epithelial cells. Ion chromatograph and confocal laser scanning microscopy were used to measure the adhesive effect between COM crystal and HK-2cells. Results The expression of hyaluronan, CD44and hyaluronan synthase1-3were higher in kidney stone group by immunohistochemistry. At the24h after exposure to calcium oxalate monohydrate（COM） crystal, the mRNA levels of HAS1-3and CD44were highest by RT-PCR, which diminished at the48h after exposure to COM crystals. The protein level of HAS1was highest at48h after exposure to COM crystals. It could be blocked by the HAS siRNA. The content of hyaluronan were measured at different time course （Oh,12h,24h,36h,48h） by ELISA kit. It began to increase at12h and reached its peak at24h. At36h after exposure it began to decrease. The adhesive effect between COM crystal and HK-2cells were reduced by the use of HAS1siRNA through confocal laser scanning microscopy and ion chromatagraph. Conclusion The expression of hyaluronan, CD44and hyaluronan synthase1-3were higher in kidney stone patients. Hyaluronan plays a crucial role in the formation of kidney stone. The adhesion of between COM crystal and HK-2cells can be decreased by the use of HAS1siRNA. Objective long noncoding RNAs （IncRNAs） are very important in biological functions. Microarray was used to reveal the changes of HK-2cells induced by COM crystals, so as to provide candidate IncRNAs involved in the molecular mechanisms concerning kidney stone formation.Methods HK-2cells were oxposed to COM crystals（lmmol/L） for24h. The microarray was used to detect the change of lncRNAs. RT-qPCR was used to validate the results.Results From the data we found there were2971lncRNAs that differentially expressed in HK-2cells exposed to lmmol/L COM crystals.1630lncRNAs were upregulated and1341lncRNAs were downregulated. Four lncRNAs （ENST00000430583、ENST00000428930、 NR₀29401and chr13） validated by qPCR were matched the result of microarray.Conclusion Our study is the first one to determine lncRNAs expression patterns in HK-2cells exposed to COM crystals. The results displayed that clusters of lncRNAs were aberrantly expressed may exert a partial role in stone formation. Taken together, this study may provide potential targets for future treatment of stone disease and novel insights into stone formation biology.更多还原