【作者】 汤进；

【导师】 蒋先镇；

【作者基本信息】 中南大学 ， 临床医学， 2013， 博士

【摘要】 目的：观察ADO信号通路对肾间质纤维化过程的影响。方法：(1)动物分组：共44只雄性小鼠,采取随机分组的方式,分成3个组别：其中,假手术组(Sham)12只,模型组(UUO)16只,干预组(PT)16只。干预组在单侧输尿管结扎(UUO)基础上予茶碱(8-PT)10mg/kg·d腹腔注射(1次/天),模型组在UUO基础上予生理盐水腹腔注射。于实验第1、3、7、14天分批次对小鼠进行处死操作,其中,假手术组实验小鼠,每次处死只数为3只,而模型组和干预组这两个组别的小鼠,每次处死的只数为4只。在处死前24小时将小鼠置入代谢笼内收集24小时尿测定尿肌酐(UCr)、β2微球蛋白(β2-MG)含量；处死前1.5小时通过小鼠阴茎静脉注射低氧探针Hypoxy probe-1(60mg/kg)。小鼠处死后,留取进行了结扎处理一侧的肾组织,开展后续的形态学以及免疫组织化学两个方面的研究。(2)观察指标：通过对肾组织进行HE以及Masson染色处理后,观察三个不同实验组的小鼠肾组织中肾小管受损的程度、肾间质中炎性细胞的侵润状况以及肾间质纤维化程度；运用免疫组织化学技术观察实验对象的肾小管上皮细胞和间质细胞的增殖细胞核抗原(PCNA)表达状况以及肾组织低氧程度及分布；利用高效液相色谱法(HPLC)检测肾组织中腺苷(ADO)的水平。结果：(1)UUO术后肾组织处于持续低氧状态,肾组织内ADO水平较假手术组明显增高；(2)UUO术后小鼠肾组织均发生了不同程度的肾小管受损状况,而茶碱能够对这类损伤提供一定的保护作用；(3)在实验小鼠肾间质出现纤维化的过程中,PCNA的阳性表达随时间延续主要集中于肾间质部分；尤其是UUO组,肾间质细胞持续处于高增殖状态；(4)在模型组实验小鼠的观察过程中发现：小鼠的肾间质炎性细胞侵润随病程的进展出现了进行性加重的趋势,使用茶碱进行处理后这种炎性细胞侵润的状况则大幅降低；(5)使用茶碱处理后,干预组小鼠的肾间质纤维化程度显著低于同期模型组。结论：(1)肾间质纤维化过程中存在肾组织持续低氧,伴随组织内ADO水平升高；(2)阻断ADO信号通路,能抑制炎症细胞侵润、肾间质细胞增殖及纤维化,对肾功能起保护作用。图12幅,参考文献39篇。目的：观察肾间质纤维化过程中相关致纤维化因子的动态变化与ADO的关系。进一步阐明肾间质纤维化的发生机制和ADO致纤维化的分子机制。方法：44只雄性小鼠随机分成3组：假手术组(Sham)12只,模型组(UUO)16只,干预组(PT)16只。干预组在单侧输尿管结扎(UUO)基础上予茶碱(8-PT)10mg/kg·d腹腔注射(1次/天),模型组在UUO基础上予生理盐水腹腔注射。利用RT-PCR方法动态观察术后第1、3、7、14天肾组织TGF-β1、PAI-1、α1(Ⅰ) procollagen mRNA表达；并利用免疫组织化学方法观察各实验组肾组织TGF-β1、α平滑肌肌动蛋白(α-SMA)的蛋白表达和分布规律。结果：(1)模型组小鼠肾组织TGF-β1、 PAI-1、α1(Ⅰ) procollagen mRNA表达明显上调并随时间延续进行性升高。(2)阻断ADO信号通路后对肾组织TGF-β1、PAI-1、 α1(Ⅰ) procollagen mRNA表达均具有显著抑制作用。(3)模型组小鼠肾组织TGF-β1、α-SMA蛋白水平表达较假手术组在各时间段均显著上调；阻断ADO通路后,上述细胞因子的表达受到明显抑制。结论：(1)腺苷信号通路通过调节细胞因子TGF-β1、PAI-1、α1(Ⅰ) procollagen mRNA表达,干预肾间质纤维化的进展。(2)ADO信号通路能调节成纤维细胞的活化以及转分化的过程。图13幅,表5幅,参考文献17篇。目的：观察ADO信号通路对小鼠肾成纤维细胞生物学行为的影响。方法：小鼠肾成纤维细胞株NIH3T3,采用TaqMan探针法确定成纤维细胞表面腺苷受体的类型。再将细胞株分为4组：对照组；NECA组(予以NECA); PT组(予以NECA+8-PT); MRS组(予以NECA+MRS1754),动态观察药物干预第1、2、3天后细胞内TGF-β1、α1(Ⅰ)procollagen、α-SMA mRNA的表达水平。运用MTT法,测定以上4组细胞在实验0、12、24、48、72小时的增殖情况。结果：(1)肾成纤维细胞表面以A2BR为主要ADO受体类型；(2) NECA刺激能上调TGF-β1、α1(Ⅰ)procollagen、α-SMA mRNA的表达,而8-PT与MRS1754则能显著抑制TGF-β1、 a1(Ⅰ)procollagen、α-SMA mRNA的表达；(3) NECA能促进成纤维细胞增殖,而8-PT与MRS1754则能抑制成纤维细胞的增殖。结论：(1)腺苷通过成纤维细胞表面A2BR介导,上调细胞内促纤维化细胞因子以及细胞增殖活化,导致纤维化发生和进展(2)A2BR阻断剂能有效抑制成纤维细胞增殖活化及促纤维化细胞因子的生成。图27幅,表15幅,参考文献24篇。更多还原

【Abstract】 Purpose:To observe the influence of the ADO signal pathway on the renal interstitial fibrosis.Methods:(1) Animals grouping:44male mice were randomly divided into3groups, including sham-operation group (n=12), model group (n=16) and intervention group (n=16). The intervention group was given theophylline and10mg/kg·d intraperitoneal injection (once/day) on the basis of the unilateral ureteral ligation and the model group was given the normal saline intraperitoneal injection based on the UUO. The mice were sacrificed respectively in the1st,3rd,7th and14th days of the experiment, among which three mice were killed in the sham-operation group at each time, and four mice were respectively killed in the model group and the intervention group at each time. The mice were put into the metabolism cage within24hours before being killed to collect the urine so that the content of the urine creatinine and the β2-microglobulin could be determined; the hypoxia probe was injected into the mouse penis vein within1.5hours before being killed. After the mice were killed, the renal tissues of the ligation side were taken for chemical study of the morphology and the immunologic tissue.(2) Observation target:After the renal tissue was stained by HE and Masson, it was observed for the renal tubular injury, the renal interstitial inflammatory cell infiltration and the interstitial fibrosis degree of the each experimental group; the immunohistochemistry technique is used to observe the epithelial cells of the renal tubular injury, proliferation cell nuclear antigen expression of the interstitial cells and the hypoxia degree and distribution of the renal tissues and the HPLC is adopted to detect the adenosine level in the renal tissue.Results:(1) Renal tissues was in the continued hypoxia status after UUO operation and the ADO level in the renal tissues was notably higher than that in the sham-operation group;(2) Broad renal tubular injury occurred after UUO and the8-PT had a protective effect on the renal tubular injury;(3) PCNA positive expression along with the time progress mainly focused on renal interstitial part in the renal interstitial fibrosis process, especially the UUO group, while the renal interstitial cells remained in the high proliferative status;(4) the renal interstitial inflammatory cell infiltration of the model group increased progressively along with the disease course progress and its inflammatory cell infiltration decreased significantly after8-PT intervention;(5) the renal interstitial fibrosis degree of the intervention group was significantly lower than that of the model group in the same period.Conclusion:(1) Hypoxia status of renal tissues exists in the renal interstitial fibrosis process and increases along with ADO level in the tissues;(2) Blocking the ADO signal pathway could restrain inflammatory cell infiltration, renal interstitial cell proliferation and fibrosis, and also has a protective effect on the renal function. Purpose:To observe the relationship between dynamic changes of the fibrogenic cytokines leading to fibrosis and the ADO in the renal interstitial fibrosis process, and to further elucidate the renal interstitial fibrosis mechanism and ADO molecular mechanism leading to the fibrosis.Methods:The RT-PCR is used to dynamically observe the TGF-β1, PAI-1and α1(I) procollagen mRNA expression of the renal tissues in the1st, the3rd, the7th and the14th days after operation; immunohistochemical method is adopted to observe the protein expression and distribution regulation of TGF-β1and a-SMA of renal tissues of each experimental group.Results:(1) The expression of renal tissue cytokines of the mice in the model group raised up and progressively increased along with the extension of the time.(2) The blocking-up of the ADO pathway had a significant inhibition function on the expression of the renal tissue cytokines.(3) The expressions of TGF-β1and a-SMA protein levels of the mouse renal tissue in the model group were significantly up-regulated in various experimental periods; after the ADO pathway was blocked-up, these cytokines’ expressions were obviously inhibited. Conclusion:(1) adenosine signal pathway interferes the progress of the renal interstitial fibrosis by adjusting the cytokines expression.(2) ADO signal pathway could regulate activation and transdifferentiation process of the fibroblast. Purpose:To study the biological behaviour change of the mouse renal fibroblast in different intervention condition.Methods:For the mouse renal fibroblast strain NIH3T3, the TaqMan probe method is used to identify the adenosine receptor type of the fibroblast surface. Four groups were divided:control group; NECA group (NECA); PT group (NECA+8-PT); MRS group (NECA+MRS1754). It was dynamically observed for TGF-β1, α1(Ⅰ)procollagen and α-SMA mRNA expression levels in the cells after the1st, the2nd and the3rd days of the drug intervention. The MMT methods are used to determine the proliferation condition above four groups of cells after being experimented for0,12,24,48and72hours.Results:The main receptor type of the renal fibroblast surface was A2BR;(2) NECA stimulation could up-regulate TGF-β1,α1(Ⅰ)procollagen and a-SMA mRNA expressions, and8-PT and MRS1754could significantly restrain TGF-β1,α1(Ⅰ)procollagen and α-SMA mRNA expressions;(3) NECA could promote the fibroblast proliferation, and8-PT and MRS1754could restrain the fibroblast proliferation.Conclusion:(1) Adenosine up-regulates pro-fibrogenic cytokines and cell proliferation activation in the cells through A2BR inducing on the fibroblast surface, leading to fibrosis occurrence and progress;(2) A2BR blocking agent could effectively restrain proliferation and activation of the fibroblast and the generation of the pro-fibrogenic cytokines.更多还原