巨噬细胞参与肾乳头钙化斑介导草酸钙结石形成的调控作用

发布时间：2018-06-04 12:54

本文选题：肾乳头钙化斑 + 草酸钙肾结石　；参考：《广西医科大学》2015年博士论文

【摘要】：第一部分：巨噬细胞和HMGB1在肾乳头钙化斑组织中的表达和作用分析目的：探讨巨噬细胞和HMGB1在肾乳头钙化斑介导草酸钙结石形成中的作用。方法：收集13例草酸钙肾结石患者的肾乳头钙化组织标本作为实验组,同期12例行根治性肾切除术的肾肿瘤患者正常肾乳头组织标本作为对照组。通过RT-PCR和免疫组化技术检测HMGB1和巨噬细胞特异性标记物CD68在肾乳头组织中的表达和分布情况,并利用透射电子显微镜观察肾乳头钙化组织中巨噬细胞、肾小管上皮细胞与草酸钙晶体的分布情况。结果：RT-PCR和免疫组化技术检测的结果提示实验组中CD68和HMGB1的表达水平较对照组高。巨噬细胞主要表达于肾间质和小血管腔,而HMGB1主要表达于肾小管上皮细胞和肾间质。电镜观察到肾乳头钙化组织间质中存在巨噬细胞的浸润和巨噬细胞吞噬草酸钙晶体的现象。结论：巨噬细胞、HMGB1可能参与了肾乳头钙化斑介导草酸钙结石的形成过程。第二部分：巨噬细胞与一水草酸钙晶体相互作用的体外研究目的：观察巨噬细胞与一水草酸钙(calcium oxalate monohydrate, COM)晶体的相互作用和探讨COM晶体刺激巨噬细胞HMGB1的表达规律。方法：在相差显微镜下观察COM晶体刺激巨噬细胞后的细胞形态变化；利用活细胞成像技术观察巨噬细胞与COM晶体相互作用的过程。用100ug/ml COM晶体刺激巨噬细胞,在刺激后0、6、12、24h和36h, RT-PCR技术检测细胞中HMGB1 mRNA的表达情况；用Western blot技术检测细胞总蛋白和细胞浆内HMGB1蛋白的表达情况；分别于COM晶体刺激后0、1、2h和4h,用ELISA技术检测细胞培养液上清中TNF-a和IL-6的含量。结果：1、通过活细胞成像观察到巨噬细胞通过粘附和胞饮对一水草酸钙晶体进行吞噬和消化,并对晶体起到转运作用。2、RT-PCR结果显示,COM晶体刺激后0-12h,培养细胞中HMGB1的mRNA表达量无明显变化,刺激后18-24h培养细胞中HMGB1的mRNA表达量明显增加。COM晶体刺激0-6h后,巨噬细胞浆内HMGB1蛋白的含量较低,12-36h后,细胞浆内HMGB1蛋白逐渐增加。COM晶体刺激后0-6h,巨噬细胞总蛋白的HMGB1蛋白含量不高,在刺激后12h细胞总蛋白HMGB1的含量开始增加,并且在刺激后24-36h保持在较高水平。3、ELISA结果显示,COM晶体刺激2h后,细胞培养液上清中TNF-a和IL-6表达增加,4h达到明显高峰。结论：1、巨噬细胞具有吞噬和清除COM晶体的能力,这可能是机体对晶体在肾脏的沉积而启动的一种免疫保护性机制。2、COM晶体可以诱导巨噬细胞HMGB1、TNF-α和IL-6炎症因子的表达；HMGB1的分泌时间明显晚于TNF-α和IL-6,这可能是晶体诱导肾脏慢性炎症损伤的重要机制之一。第三部分：磷酸钙晶体刺激肾小管上皮细胞HMGB1表达的实验研究目的：探讨磷酸钙晶体刺激人肾小管上皮细胞(HK-2) HMGB1mRNA和蛋白表达水平。方法：利用相差显微镜观察磷酸钙晶体刺激后肾小管上皮细胞的形态变化；用100ug/ml磷酸钙晶体刺激肾小管上皮细胞18h、24h、30h,用R-T PCR技术检测细胞HMGB1mRNA相对表达量；用100ug/ml磷酸钙晶体刺激肾小管上皮细胞0、3、6、9、12h、18h、24h和48h,用Western blot技术检测细胞总蛋白中HMGB1的含量。结果：1、磷酸钙晶体刺激肾小管上皮细胞18h后,HMGB1mRNA表达开始上升,至30h达到高峰,组间的差异具有统计学意义(p0.05).2、磷酸钙晶体刺激肾小管上皮细胞3h、6h、9h后,HMGB1蛋白表达水平与0h间的差异无统计学意义,但18-48h呈现时间依赖性增加,48小时达到高峰,与0h相比,具有统计学意义(p0.05)。结论：磷酸钙晶体刺激肾小管上皮细胞HMGB1持续表达的时间长,从而造成肾乳头慢性炎性损伤,这可能是炎症介导肾乳头钙化斑形成的重要原因之一。第四部分：HMGB1对磷酸钙晶体诱导巨噬细胞释放炎症因子的协同效应研究目的：探讨HMGB1对磷酸钙晶体诱导巨噬细胞释放IL-1β、IL-6、TNF-α、MCP-1早期炎症因子的协同作用及其可能机制。方法：1、将巨噬细胞分为空白组、100ug/mlCaP组、100ng/mlHMGB1组、100ug/mlCaP+100ng/mlHMGB1组,干预1、2、4h后收集细胞上清液,ELISA法检测IL-1β、IL-6、TNF-α、MCP-1含量2、将100ug/mlCaP+不同浓度HMGB1(10ng/ml,50ng/ml,100ng/ml)干预巨噬细胞4h后收集细胞上清液,ELISA法检测IL-1β、IL-6、TNF-α、MCP-1含量；3、将100ug/mlCaP干预巨噬细胞1,2,4h, RT-PCR技术检测NF-KB mRNA表达水平；4、将100ug/mlCaP+不同浓度HMGB1(100ng/ml,500ng/ml,1000ng/ml)干预巨噬细胞1h, RT-PCR技术检测NF-KB mRNA表达水平。结果：1、ELISA结果显示100ug/mlCaP组,100ng/mlHMGB1组上清液IL-1β、IL-6、TNF-α、MCP-1含量均高于空白组,100ug/mlCaP+100ng/mlHMGBl组上清液IL-1β、IL-6. TNF-α、MCP-1含量均高于空白组、100ug/mlCaP组、100ng/mlHMGB1组(P0.05),且呈时间依赖性；2、不同浓度HMGBl+100ug/mlCaP组细胞上清液IL-1β、IL-6、TNF-α、MCP-1含量均高于100ug/mlCaP 组(P0.05),且呈浓度依赖性。3、100 μg/mlCaP刺激巨噬细胞1、2、4h,与对照组比较,3组巨噬细胞NF-KB mRNA目对表达量均升高,具有时间依赖性,且4h达高峰(p0.05)。100μg/mlCaP+不同浓度HMGB1组的巨噬细胞NF-KBmRNA相对表达量较100μg/mlCaP均升高(p0.05),且呈浓度依赖性。结论：1. HMGB1可协同CaP诱导巨噬细胞释放IL-1β、IL-6、TNF-α、MCP-1炎症因子；2.HMGB1协同CaP诱导巨噬细胞释放早期炎症因子可能与NF-KB信号通路激活有关。第五部分：巨噬细胞在磷酸钙—肾小管上皮细胞体系中的调控作用研究目的：在磷酸钙-巨噬细胞、肾小管上皮细胞的共培养体系中,探讨磷酸钙晶体对巨噬细胞体外迁移作用的影响以及巨噬细胞对该体系中TGF-p、MCP-1炎症因子的调控作用。方法：采用transwell小室分层非接触的共培养系统,A组上层小室接种巨噬细胞,下层接种肾小管上皮细胞,B组上层小室接种巨噬细胞,下层接种肾小管上皮细胞和添加磷酸钙,观察两组不同时间点巨噬细胞的迁移情况。C组单纯下层小室接种肾小管上皮细胞和添加磷酸钙。D组单纯下层小室接种肾小管上皮细胞。采用ELISA技术检测B,C,D组的细胞培养液上清MCP-1、TGF-β浓度。RT-PCR技术检测B,C,D组肾小管上皮细胞MCP-1、TGF-βmRNA表达水平。结果：B组在磷酸钙晶体刺激6h和9h后,与A组相比,巨噬细胞迁移的数量逐渐增多(P0.05),在磷酸钙晶体刺激12h后,两组间的巨噬细胞迁移数量无明显差异(P0.05)。ELISA检测发现,B组培养液上清中的MCP-1、TGF-β浓度分别在1,3,6h和24,48,72h与C、D组相比明显升高(P0.05)。RT-PCR结果显示B组肾小管上皮细胞TGF-β、MCP-1mRNA表达水平分别在1,3,6h和24,48,72h与C、D组相比也明显升高(P0.05)。结论：1、磷酸钙晶体对巨噬细胞迁移具有促进作用,其作用可能与磷酸钙晶体刺激肾小管上皮细胞表达MCP-1有关。2、巨噬细胞对磷酸钙-肾小管上皮细胞反应体系中的MCP-1、TGF-β炎症因子释放具有明显的促进作用。
[Abstract]:Part one: expression and effect of macrophage and HMGB1 in the tissue of renal papillary calcification. Objective: To explore the role of macrophage and HMGB1 in the formation of calcium oxalate calcium lithiasis mediated by renal papillary calcification. Methods: 13 cases of calcium oxalate nephrolithiasis were collected as experimental group, and 12 cases were treated with radical cure in the same period. The normal papillary tissue specimens of renal tumor patients with nephrectomy were used as the control group. The expression and distribution of HMGB1 and macrophage specific marker CD68 in the renal papilla tissue were detected by RT-PCR and immunohistochemistry, and the macrophages, renal tubular epithelial cells and renal tubular cells were observed by transmission electron microscopy. The distribution of calcium oxalate crystals. Results: the results of RT-PCR and immunohistochemistry showed that the expression level of CD68 and HMGB1 in the experimental group was higher than that of the control group. The macrophages were mainly expressed in the renal interstitium and the small vascular cavity, while HMGB1 was mainly expressed in the renal tubular epithelial cells and the renal interstitium. There is a phenomenon of macrophage infiltration and macrophage phagocytosis of calcium oxalate crystals. Conclusion: macrophages, HMGB1 may be involved in the formation of calcium oxalate calcium calculus in renal papillary calcification. The second part: macrophage and calcium oxalate crystal interaction in vitro: macrophage and calcium oxalate (calci) The interaction between um oxalate monohydrate, COM) crystal and the expression of HMGB1 in macrophages stimulated by COM crystal. Methods: the morphological changes of macrophages after macrophages stimulated by COM crystal were observed under phase contrast microscope, and the interaction between macrophages and COM crystals was observed by living cell imaging technique. 100ug/ml COM crystal stings were used. The expression of HMGB1 mRNA in the cells was detected by 0,6,12,24h, 36h and RT-PCR, and the expression of the total protein and intracellular HMGB1 protein in the cytoplasm was detected by Western blot, and 0,1,2h and 4h after the COM crystal was stimulated respectively. The results were detected by ELISA technique. 1, through living cell imaging, it was observed that macrophages phagocytic and digested by adhesion and drinking on calcium oxalate crystal, and transport the crystal to the crystal.2. RT-PCR results showed that the mRNA expression of HMGB1 in the cultured cells was not obviously changed after the stimulation of COM crystal, and the mRNA expression of HMGB1 in the cultured cells of 18-24h was obvious after stimulation. After the stimulation of.COM crystal, the content of HMGB1 protein in the macrophage pulp was low. After 12-36h, the HMGB1 protein in the cytoplasm gradually increased after the.COM crystal stimulated 0-6h, and the HMGB1 protein content of the total protein of macrophage was not high. The content of the 12h cell total protein HMGB1 content began to increase after the stimulation, and the content of the total protein HMGB1 in the 12h cell was increased after the stimulation, and was kept at a higher level.3 after the stimulation. ISA results showed that when COM crystal stimulated 2h, the expression of TNF-a and IL-6 increased in the supernatant of cell culture, and the 4H reached a significant peak. Conclusion: 1, macrophages have the ability to phagocytiate and clear the COM crystals, which may be an immune protective mechanism for the organism to initiate the deposition of the crystal in the kidney, and COM crystal can induce the macrophage HMGB1, TNF. The expression of alpha and IL-6 inflammatory factors; the secretion time of HMGB1 is obviously later than TNF- alpha and IL-6, which may be one of the important mechanisms of the crystal induced chronic renal inflammation. The third part: calcium phosphate crystal stimulates the expression of HMGB1 in renal tubular epithelial cells: To explore the stimulation of calcium phosphate crystal to stimulate human renal tubular epithelial cells (HK-2). HMGB1mRNA and protein expression level. Methods: the morphological changes of renal tubular epithelial cells were observed with calcium phosphate crystals by phase contrast microscope; 18h, 24h, 30h of renal tubular epithelial cells were stimulated by 100ug/ml calcium phosphate crystal. R-T PCR technique was used to detect the relative expression of cell HMGB1mRNA, and the renal tubule epithelium was stimulated with 100ug/ml phosphate crystal. Cells 0,3,6,9,12h, 18h, 24h and 48h were used to detect the content of HMGB1 in the cell total protein with Western blot. Results: 1, after calcium phosphate crystals stimulated the renal tubular epithelial cells 18h, the HMGB1mRNA expression began to rise, to the peak of 30h, and the difference between the groups was statistically significant (P0.05).2, calcium phosphate crystals stimulated the renal tubular epithelial cells. There was no statistical difference between the expression level of HMGB1 protein and 0h, but the time dependence of 18-48h increased and reached a peak at 48 hours. Compared with 0h, it was statistically significant (P0.05). Conclusion: calcium phosphate crystal stimulates the prolonged expression of HMGB1 in renal tubular epithelial cells, which may cause chronic inflammatory injury of the renal papilla, which may be an inflammation. One of the important reasons for the formation of renal papillary calcification. Fourth: the fourth part: the synergistic effect of HMGB1 on the release of inflammatory factors from macrophages in calcium phosphate crystals: To explore the synergistic effect and possible mechanism of HMGB1 on the release of IL-1 beta, IL-6, TNF- alpha and MCP-1 in macrophages by calcium phosphate crystals. Methods: 1, Macrophages were divided into blank group, group 100ug/mlCaP, group 100ng/mlHMGB1 and group 100ug/mlCaP+100ng/mlHMGB1. After intervention 1,2,4h, cell supernatant was collected. IL-1 beta, IL-6, TNF- a, MCP-1 content was detected by ELISA method, and MCP-1 content was 2. 6, TNF- alpha, MCP-1 content; 3, 100ug/mlCaP intervention macrophage 1,2,4h, RT-PCR technology to detect NF-KB mRNA expression level. 4, 100ug/mlCaP+ concentration HMGB1 (100ng/ml, 500ng/ml, 1000ng/ml) intervention macrophage expression level. The content of IL-1 beta, IL-6, TNF-, and MCP-1 in the liquid was higher than that in the blank group. The content of IL-1 beta and IL-6. TNF- a in 100ug/mlCaP+100ng/mlHMGBl group was higher than that in the blank group, 100ug/mlCaP group and 100ng/mlHMGB1 group (P0.05), and was time dependent. The content of the cell supernatant of different concentrations was higher than that of the 100ug/mlCaP+100ng/mlHMGBl group. AP group (P0.05) and concentration dependent.3100 mu g/mlCaP stimulated macrophage 1,2,4h. Compared with the control group, the expression of NF-KB mRNA mesh increased in the 3 groups, which was time dependent, and the relative expression of macrophages in the HMGB1 group increased with the peak of 4H reaching the peak (P0.05).100 u g/mlCaP+, and the relative expression of the macrophage was higher than that of the 100 micron. Conclusion: 1. HMGB1 can synergistically induce macrophage to release IL-1 beta, IL-6, TNF- alpha, MCP-1 inflammatory factors. The release of early inflammatory factors by 2.HMGB1 synergistic CaP in macrophages may be related to the activation of NF-KB signaling pathway. The fifth part: the role of macrophages in the regulatory role of macrophages in the calcium phosphate renal tubular epithelial cell system. Objective: in the co culture system of calcium phosphate macrophages and renal tubular epithelial cells, the effect of calcium phosphate on the migration of macrophages in vitro and the regulation of macrophage on TGF-p and MCP-1 inflammatory factors in this system. Methods: the non contact co culture system of Transwell compartment and the upper layer of the upper layer of group A were used. The macrophage and lower layer were inoculated with renal tubular epithelial cells, the B group was inoculated with macrophages in the upper chamber, the lower layer was inoculated with renal tubular epithelial cells and calcium phosphate was added. The migration of macrophages in two groups at different time points was observed. Group.C was inoculated with renal tubular epithelial cells and calcium phosphate.D group to inoculate renal tubules alone. Epithelial cells. The cell culture fluid of B, C and D group was detected by ELISA technique, MCP-1, TGF- beta concentration.RT-PCR technique was used to detect B, C, D group renal tubular epithelial cells MCP-1, TGF- beta expression level. After that, there was no significant difference in the number of macrophage migration between the two groups (P0.05).ELISA detection. The concentration of MCP-1 in the supernatant of group B culture, TGF- beta concentration was significantly higher in 1,3,6h and 24,48,72h than in C and D group (P0.05).RT-PCR results showed that the renal tubular epithelial cells of the B group were compared with those of the group. It is also significantly increased (P0.05). Conclusion: 1, calcium phosphate crystals can promote the migration of macrophages. The effect of calcium phosphate crystals may be related to the expression of MCP-1.2 in renal tubular epithelial cells. Macrophages have a significant effect on the release of MCP-1 in calcium phosphate renal tubular epithelial cell response system and the release of TGF- beta inflammatory factors.
【学位授予单位】：广西医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R692.4

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