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  本文選題：腎乳頭鈣化斑 + 草酸鈣腎結(jié)石�。� 參考：《廣西醫(yī)科大學(xué)》2015年博士論文

【摘要】：第一部分：巨噬細(xì)胞和HMGB1在腎乳頭鈣化斑組織中的表達(dá)和作用分析目的：探討巨噬細(xì)胞和HMGB1在腎乳頭鈣化斑介導(dǎo)草酸鈣結(jié)石形成中的作用。方法：收集13例草酸鈣腎結(jié)石患者的腎乳頭鈣化組織標(biāo)本作為實驗組,同期12例行根治性腎切除術(shù)的腎腫瘤患者正常腎乳頭組織標(biāo)本作為對照組。通過RT-PCR和免疫組化技術(shù)檢測HMGB1和巨噬細(xì)胞特異性標(biāo)記物CD68在腎乳頭組織中的表達(dá)和分布情況,并利用透射電子顯微鏡觀察腎乳頭鈣化組織中巨噬細(xì)胞、腎小管上皮細(xì)胞與草酸鈣晶體的分布情況。結(jié)果：RT-PCR和免疫組化技術(shù)檢測的結(jié)果提示實驗組中CD68和HMGB1的表達(dá)水平較對照組高。巨噬細(xì)胞主要表達(dá)于腎間質(zhì)和小血管腔,而HMGB1主要表達(dá)于腎小管上皮細(xì)胞和腎間質(zhì)。電鏡觀察到腎乳頭鈣化組織間質(zhì)中存在巨噬細(xì)胞的浸潤和巨噬細(xì)胞吞噬草酸鈣晶體的現(xiàn)象。結(jié)論：巨噬細(xì)胞、HMGB1可能參與了腎乳頭鈣化斑介導(dǎo)草酸鈣結(jié)石的形成過程。第二部分：巨噬細(xì)胞與一水草酸鈣晶體相互作用的體外研究目的：觀察巨噬細(xì)胞與一水草酸鈣(calcium oxalate monohydrate, COM)晶體的相互作用和探討COM晶體刺激巨噬細(xì)胞HMGB1的表達(dá)規(guī)律。方法：在相差顯微鏡下觀察COM晶體刺激巨噬細(xì)胞后的細(xì)胞形態(tài)變化；利用活細(xì)胞成像技術(shù)觀察巨噬細(xì)胞與COM晶體相互作用的過程。用100ug/ml COM晶體刺激巨噬細(xì)胞,在刺激后0、6、12、24h和36h, RT-PCR技術(shù)檢測細(xì)胞中HMGB1 mRNA的表達(dá)情況；用Western blot技術(shù)檢測細(xì)胞總蛋白和細(xì)胞漿內(nèi)HMGB1蛋白的表達(dá)情況；分別于COM晶體刺激后0、1、2h和4h,用ELISA技術(shù)檢測細(xì)胞培養(yǎng)液上清中TNF-a和IL-6的含量。結(jié)果：1、通過活細(xì)胞成像觀察到巨噬細(xì)胞通過粘附和胞飲對一水草酸鈣晶體進(jìn)行吞噬和消化,并對晶體起到轉(zhuǎn)運作用。2、RT-PCR結(jié)果顯示,COM晶體刺激后0-12h,培養(yǎng)細(xì)胞中HMGB1的mRNA表達(dá)量無明顯變化,刺激后18-24h培養(yǎng)細(xì)胞中HMGB1的mRNA表達(dá)量明顯增加。COM晶體刺激0-6h后,巨噬細(xì)胞漿內(nèi)HMGB1蛋白的含量較低,12-36h后,細(xì)胞漿內(nèi)HMGB1蛋白逐漸增加。COM晶體刺激后0-6h,巨噬細(xì)胞總蛋白的HMGB1蛋白含量不高,在刺激后12h細(xì)胞總蛋白HMGB1的含量開始增加,并且在刺激后24-36h保持在較高水平。3、ELISA結(jié)果顯示,COM晶體刺激2h后,細(xì)胞培養(yǎng)液上清中TNF-a和IL-6表達(dá)增加,4h達(dá)到明顯高峰。結(jié)論：1、巨噬細(xì)胞具有吞噬和清除COM晶體的能力,這可能是機(jī)體對晶體在腎臟的沉積而啟動的一種免疫保護(hù)性機(jī)制。2、COM晶體可以誘導(dǎo)巨噬細(xì)胞HMGB1、TNF-α和IL-6炎癥因子的表達(dá)；HMGB1的分泌時間明顯晚于TNF-α和IL-6,這可能是晶體誘導(dǎo)腎臟慢性炎癥損傷的重要機(jī)制之一。第三部分：磷酸鈣晶體刺激腎小管上皮細(xì)胞HMGB1表達(dá)的實驗研究目的：探討磷酸鈣晶體刺激人腎小管上皮細(xì)胞(HK-2) HMGB1mRNA和蛋白表達(dá)水平。方法：利用相差顯微鏡觀察磷酸鈣晶體刺激后腎小管上皮細(xì)胞的形態(tài)變化；用100ug/ml磷酸鈣晶體刺激腎小管上皮細(xì)胞18h、24h、30h,用R-T PCR技術(shù)檢測細(xì)胞HMGB1mRNA相對表達(dá)量；用100ug/ml磷酸鈣晶體刺激腎小管上皮細(xì)胞0、3、6、9、12h、18h、24h和48h,用Western blot技術(shù)檢測細(xì)胞總蛋白中HMGB1的含量。結(jié)果：1、磷酸鈣晶體刺激腎小管上皮細(xì)胞18h后,HMGB1mRNA表達(dá)開始上升,至30h達(dá)到高峰,組間的差異具有統(tǒng)計學(xué)意義(p0.05).2、磷酸鈣晶體刺激腎小管上皮細(xì)胞3h、6h、9h后,HMGB1蛋白表達(dá)水平與0h間的差異無統(tǒng)計學(xué)意義,但18-48h呈現(xiàn)時間依賴性增加,48小時達(dá)到高峰,與0h相比,具有統(tǒng)計學(xué)意義(p0.05)。結(jié)論：磷酸鈣晶體刺激腎小管上皮細(xì)胞HMGB1持續(xù)表達(dá)的時間長,從而造成腎乳頭慢性炎性損傷,這可能是炎癥介導(dǎo)腎乳頭鈣化斑形成的重要原因之一。第四部分：HMGB1對磷酸鈣晶體誘導(dǎo)巨噬細(xì)胞釋放炎癥因子的協(xié)同效應(yīng)研究目的：探討HMGB1對磷酸鈣晶體誘導(dǎo)巨噬細(xì)胞釋放IL-1β、IL-6、TNF-α、MCP-1早期炎癥因子的協(xié)同作用及其可能機(jī)制。方法：1、將巨噬細(xì)胞分為空白組、100ug/mlCaP組、100ng/mlHMGB1組、100ug/mlCaP+100ng/mlHMGB1組,干預(yù)1、2、4h后收集細(xì)胞上清液,ELISA法檢測IL-1β、IL-6、TNF-α、MCP-1含量2、將100ug/mlCaP+不同濃度HMGB1(10ng/ml,50ng/ml,100ng/ml)干預(yù)巨噬細(xì)胞4h后收集細(xì)胞上清液,ELISA法檢測IL-1β、IL-6、TNF-α、MCP-1含量；3、將100ug/mlCaP干預(yù)巨噬細(xì)胞1,2,4h, RT-PCR技術(shù)檢測NF-KB mRNA表達(dá)水平；4、將100ug/mlCaP+不同濃度HMGB1(100ng/ml,500ng/ml,1000ng/ml)干預(yù)巨噬細(xì)胞1h, RT-PCR技術(shù)檢測NF-KB mRNA表達(dá)水平。結(jié)果：1、ELISA結(jié)果顯示100ug/mlCaP組,100ng/mlHMGB1組上清液IL-1β、IL-6、TNF-α、MCP-1含量均高于空白組,100ug/mlCaP+100ng/mlHMGBl組上清液IL-1β、IL-6. TNF-α、MCP-1含量均高于空白組、100ug/mlCaP組、100ng/mlHMGB1組(P0.05),且呈時間依賴性；2、不同濃度HMGBl+100ug/mlCaP組細(xì)胞上清液IL-1β、IL-6、TNF-α、MCP-1含量均高于100ug/mlCaP 組(P0.05),且呈濃度依賴性。3、100 μg/mlCaP刺激巨噬細(xì)胞1、2、4h,與對照組比較,3組巨噬細(xì)胞NF-KB mRNA目對表達(dá)量均升高,具有時間依賴性,且4h達(dá)高峰(p0.05)。100μg/mlCaP+不同濃度HMGB1組的巨噬細(xì)胞NF-KBmRNA相對表達(dá)量較100μg/mlCaP均升高(p0.05),且呈濃度依賴性。結(jié)論：1. HMGB1可協(xié)同CaP誘導(dǎo)巨噬細(xì)胞釋放IL-1β、IL-6、TNF-α、MCP-1炎癥因子；2.HMGB1協(xié)同CaP誘導(dǎo)巨噬細(xì)胞釋放早期炎癥因子可能與NF-KB信號通路激活有關(guān)。第五部分：巨噬細(xì)胞在磷酸鈣—腎小管上皮細(xì)胞體系中的調(diào)控作用研究目的：在磷酸鈣-巨噬細(xì)胞、腎小管上皮細(xì)胞的共培養(yǎng)體系中,探討磷酸鈣晶體對巨噬細(xì)胞體外遷移作用的影響以及巨噬細(xì)胞對該體系中TGF-p、MCP-1炎癥因子的調(diào)控作用。方法：采用transwell小室分層非接觸的共培養(yǎng)系統(tǒng),A組上層小室接種巨噬細(xì)胞,下層接種腎小管上皮細(xì)胞,B組上層小室接種巨噬細(xì)胞,下層接種腎小管上皮細(xì)胞和添加磷酸鈣,觀察兩組不同時間點巨噬細(xì)胞的遷移情況。C組單純下層小室接種腎小管上皮細(xì)胞和添加磷酸鈣。D組單純下層小室接種腎小管上皮細(xì)胞。采用ELISA技術(shù)檢測B,C,D組的細(xì)胞培養(yǎng)液上清MCP-1、TGF-β濃度。RT-PCR技術(shù)檢測B,C,D組腎小管上皮細(xì)胞MCP-1、TGF-βmRNA表達(dá)水平。結(jié)果：B組在磷酸鈣晶體刺激6h和9h后,與A組相比,巨噬細(xì)胞遷移的數(shù)量逐漸增多(P0.05),在磷酸鈣晶體刺激12h后,兩組間的巨噬細(xì)胞遷移數(shù)量無明顯差異(P0.05)。ELISA檢測發(fā)現(xiàn),B組培養(yǎng)液上清中的MCP-1、TGF-β濃度分別在1,3,6h和24,48,72h與C、D組相比明顯升高(P0.05)。RT-PCR結(jié)果顯示B組腎小管上皮細(xì)胞TGF-β、MCP-1mRNA表達(dá)水平分別在1,3,6h和24,48,72h與C、D組相比也明顯升高(P0.05)。結(jié)論：1、磷酸鈣晶體對巨噬細(xì)胞遷移具有促進(jìn)作用,其作用可能與磷酸鈣晶體刺激腎小管上皮細(xì)胞表達(dá)MCP-1有關(guān)。2、巨噬細(xì)胞對磷酸鈣-腎小管上皮細(xì)胞反應(yīng)體系中的MCP-1、TGF-β炎癥因子釋放具有明顯的促進(jìn)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.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R692.4

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 付亮;蔡紹曦;趙海金;李文軍;佟萬成;;N-乙酰半胱氨酸對哮喘小鼠肺組織HMGB1、RAGE表達(dá)的影響[J];南方醫(yī)科大學(xué)學(xué)報;2008年05期

2 陶芝偉;鄧耀良;黎承楊;;利用電鏡技術(shù)對尿路鈣化性納米微粒的初步觀察[J];廣西醫(yī)科大學(xué)學(xué)報;2009年06期

3 鄧耀良;Randall斑的歷史、現(xiàn)狀和未來[J];廣西醫(yī)學(xué);2005年01期

4 何林祥;孫航;吳傳新;龔建平;劉杞;郭暉;;LPS通過P38MAPK-CBP誘導(dǎo)巨噬細(xì)胞RAW264.7表達(dá)和釋放HMGB1[J];基礎(chǔ)醫(yī)學(xué)與臨床;2012年05期

5 李成文;孫光;鄧耀良;李盛寬;米華;劉德云;;草酸鈣晶體對大鼠腎小管上皮細(xì)胞損傷作用的實驗研究[J];現(xiàn)代泌尿外科雜志;2006年02期

6 魏仁波;王磊;張榮貴;林艷君;徐光勇;張唯力;;α-黑素細(xì)胞刺激素對慢性非細(xì)菌性前列腺炎大鼠HMGB1表達(dá)的影響[J];中國男科學(xué)雜志;2010年07期

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9 何登;王少剛;唐錦輝;賈招輝;郭丙濤;黃雷;盧宇超;;特發(fā)性高鈣尿腎結(jié)石患者腎乳頭組織中Runx2、Osterix的表達(dá)[J];華中科技大學(xué)學(xué)報(醫(yī)學(xué)版);2013年02期

10 桂亞平;劉博;卞崔冬;袁濤;姜啟全;;前列腺癌根治術(shù)后HMGB1表達(dá)的意義[J];同濟(jì)大學(xué)學(xué)報(醫(yī)學(xué)版);2014年03期

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