【作者】 肖红波；

【导师】 刘瑞洪；

【作者基本信息】 中南大学 ， 内科学， 2010， 博士

【摘要】 背景肾纤维化是指在各种致病因子如炎症、损伤等作用下,间质细胞及细胞间质增多,尤其是基质蛋白合成增加,基质降解受抑制造成细胞外基质(Extracellular matrix, ECM)的大量堆积导致的肾小球硬化和小管间质纤维化。它是多种慢性肾脏疾病最终导致肾功能衰竭的主要病理改变和共同通路,延缓和防止肾纤维化是防治慢性肾脏病进展的关键。胶原是ECM的主要成分,在各种肾脏纤维化疾病中合成都显著增高,它合成和沉积增加与肾脏纤维化的启动和进展有密切关系。热休克蛋白47(Heat shock protein 47, HSP47)是一种胶原特异性的“分子伴侣”,在胶原生物合成过程中协助前胶原修饰、折叠、装配,是胶原成熟的必要条件,其表达影响胶原产量。HSP47在多种肾脏纤维化的动物和临床疾病研究中包括高血压肾硬化模型大鼠,5／6肾脏切除大鼠模型,人类IgA肾病及糖尿病肾病肾组织等,表达都明显增高,且与胶原同步增加。并初步证实动物体内抑制其表达可减轻纤维化的程度,如HSP47反义寡聚脱氧核苷酸转染抗Thy-1肾小球肾炎大鼠,HSP47 siRNA转染UUO大鼠,都可以改善肾纤维化,提示HSP47在肾脏纤维化中的作用可能并不局限于调节胶原的合成。丝裂原活化蛋白激酶(Mitogen-activated protein kinase, MAPK)信号通路是介导细胞反应的重要信号系统,能将细胞外信号转导至细胞及其核内,通过保守的三级级联反应(MAPKKK-MAPKK-MAPK)激活转录因子,调节基因表达。细胞外调节蛋白激酶(extracellular signal-regulated kinase, ERK1/2)和c-Jun氨基末端激酶(c-JunNH2-terminal kinase, JNK)是它的两个主要家族成员。其中ERK1／2又被称为丝裂原活化的MAPK通路,主要功能是介导促进细胞增殖和分化；JNK主要功能是调节参与机体应激反应。体外实验证明ERK1／2,JNK信号传导通路可介导外界刺激所致的热休克蛋白合成。基于以上分析得知HSP47是胶原合成的特异性“分子伴侣”,其在肾脏纤维化中表达增高,且有促肾脏纤维化作用,但其作用机制可能不局限于对胶原合成的调控,MAPK信号传导通路可能参与调控HSP47的表达。为此,本实验开展如下的研究。目的利用单侧输尿管梗阻(unilateral ureteral obstruction, UUO)大鼠模型,初步观察HSP47在肾脏组织的表达情况,旨在探讨HSP47在肾小管间质纤维化中的作用。方法SD大鼠随机分为模型组(UUO组)和假手术组(Sham组),每组8只。采用单侧(左)输尿管结扎方法,建立梗阻性肾小管间质纤维化大鼠模型,Sham组只游离输尿管而不结扎。术后14天处死动物,取结扎侧肾脏,同时处死假手术组大鼠,取出肾组织。观察各组之间蛋白尿,血肌酐水平,运用HE, Masson染色观察肾脏组织形态学改变,采用免疫组化方法检测HSP47、胶原Ⅳ(CollagenⅣ)与纤维连接蛋白(Fibronectin, FN)表达的部位和变化。结果1.与假手术组比较,UUO模型组大鼠血肌酐水平升高,蛋白尿无显著差异。2.与假手术组比较,光镜下UUO模型组大鼠肾组织出现不同程度的肾小管扩张、间质炎症细胞浸润,间质面积增宽、部分肾小管萎缩、肾小管上皮细胞空泡样变性。3.Masson染色结果显示：与假手术组比较,UUO模型组大鼠肾组织胶原在肾小管周围、间质区显著增多。4.免疫组化结果显示：与假手术组比较,UUO模型组大鼠肾组织HSP47表达显著增加,在肾小管周围、间质区表达增加最明显；CollagenⅣ表达显著增加,在肾小管基底膜、间质区表达增加最明显；FN表达显著增加,主要表达在肾间质区。结论HSP47在UUO大鼠模型肾脏组织表达上调,且与胶原表达部位一致,提示HSP47促进肾间质纤维化,且该作用与其促进胶原蛋白的合成有关。目的研究HSP47在TGF-β1诱导HK-2细胞合成Collagen及FN等ECM蛋白过程中的作用,并检测其对PAI-1表达的影响,探讨HSP47在ECM合成与降解平衡中的角色。方法常规培养HK-2细胞,分为对照组、TGF-β1组、HSP47siRNA组,对照组为无血清DMEM组,TGF-p1组为TGF-p1诱导,HSP47siRNA组为HSP47siRNA与TGF-β1共同作用,RT-PCR检测HSP47、collagenⅣ、FN. PAI-1的mRNA表达,Western Blot检测HSP47、collagenⅣ、FN蛋白表达,ELISA检测PAI-1的蛋白表达。结果1.常规培养的HK-2可表达HSP47,不同浓度TGF-p1(0、2.5、5、10ng/ml)干预不同时间(12h、24h、48h), HSP47基因和蛋白表达都呈逐渐增高的趋势,10ng/ml TGF-β1干预HK-2细胞48h时HSP47在基因和蛋白水平上都表达最强。2.不同浓度TGF-β1 (0、5、10ng/ml)干预HK-2不同时间(12h、24h、48h), collagenⅣ、FN、PAI-1在基因,蛋白水平表达呈逐渐增高的趋势,TGF-β1(10ng/ml)作用48小时三者基因和蛋白水平表达最强。3.HSP47siRNA组(HSP47siRNA处理24h,再TGF-β1 10ng/ml干预48)与TGF-β1 10ng/ml干预48h比较,前者HSP47, collagenⅣ, FN, PAI-1 mRNA和蛋白的表达都明显下调。结论1.TGF-β1呈浓度和时间依赖模式上调HK-2细胞HSP47的表达。2.TGF-β1呈浓度和时间依赖模式上调HK-2细胞collagen IV, FN,PAI-1表达。3.HSP47siRNA可以下调TGF-β1诱导下HK-2细胞分泌的collagenⅣ、FN、PAI-1的mRNA和蛋白表达。目的研究丝裂原激活蛋白激酶(MAPK)途径是否介导了TGF-β1诱导HK-2细胞上调HSP47表达。方法应用ERK、JNK特异性抑制剂观察对TGF-p1诱导HK-2细胞上调HSP47表达的影响结果应用PD98059、SP600125分别特异性抑制MAPK途径的胞外信号调节蛋白激酶(ERK1/2)、c-Jun-氨基末端激酶(JNK)通路,对TGF-β1上调HSP47的作用有不同影响。1.PD98059可以抑制TGF-β1对HK-2细胞ERK信号通路的活化和HSP47的表达,且不同浓度PD98059 (25uM、50uM、75uM)对HSP47的抑制效果有差异。2.SP600125可以抑制TGF-β1对HK-2细胞JNK信号通路的活化和HSP47的表达,且不同浓度SP600125 (10uM、20uM、30uM)对HSP47的抑制效果无明显差异。结论ERK1/2、JNK信号通路参与介导TGF-p1上调HK-2细胞表达HSP47。更多还原

【Abstract】 Renal fibrosis refers to the increase of interstitial cells and intercellular matrix due to pathogenic factors such as inflammation, injury, etc, particularly, the increase of matrix protein synthesis, the inhibition of matrix degradation, leading to a huge accumulation of extracellular matrix (ECM), and leading to glomerular sclerosis and tubulointerstitial fibrosis. Ultimately, it is the main pathological changes and common pathways leading to renal failure in a number of chronic kidney diseases. Prevention and delaying of renal fibrosis is the key to preventing and arresting the progression of chronic kidney disease. Collagen is the main component of ECM, it increases significantly in various renal fibrosis diseases, and its increased synthesis and deposition are closely related to renal fibrosis.Heat shock protein 47 is a collagen-specific "molecular chaperone". During the biosynthesis of collagen, it helps in modification, folding and assembly of procollagen. It is the necessary condition for the biosynthesis of collagen, and its expression is closely related to the collagen production.The expression of HSP47 is increased significantly in a variety of animal studies and clinical diseases of renal fibrosis, including hypertensive nephrosclerosis in rats,5/6 nephrectomy rat model, human IgA nephropathy and diabetic nephropathy, etc, and the increase is directly proportional with that of collagen. Some researches preliminarily confirmed that inhibiting its expression in animals could reduce the degree of fibrosis, for example, transfecting HSP47 antisense oligonucleotides to anti-Thy-1 glomerulonephritis rats and HSP47siRNA to UUO rats could improve renal fibrosis, suggesting that its role in renal fibrosis may not be limited to regulating collagen synthesis.Mitogen-activated protein kinase (Mitogen-activated protein kinase, MAPK) signaling pathway is an important signaling system mediated cell response, which can transmit extracellular signals to the cell and its nucleus through the conservative cascade 3 (MAPKKK-MAPKK-MAPK) activated transcription factors to regulate gene expression. Extracellular regulated protein kinase (extracellular signal-regulated kinase, ERK1/2) and c-Jun N-terminal kinase (c-Jun NH2-terminal kinase, JNK) are two major members of the MAPK family, in which ERK1/2 is known as the mitogen-activated MAPK pathway. Its main function is to mediate the promotion of cell proliferation and differentiation; and the main function of JNK is to participate and regulate body stress, inflammation reaction. In-vitro researches show that the MAPK signaling pathway can mediate the synthesis of heat shock protein induced by external stimuli.The analysis above indicated that HSP47 is a collagen-specific "molecular chaperone", its expression is increased in renal fibrosis, and it could promote renal fibrosis, its role and mechanism in renal fibrosis may not be limited to the regulation of collagen synthesis alone. MAPK signaling pathway may also be involved in the regulation of synthesis of HSP47. To this end, the following research was carried out. Objective:To observe the expression of HSP47 in renal tissues of UUO rats and investigate its role in renal interstitial fibrosis.Method:Sprague-Dawley rats were randomly divided into control and UUO groups (each group:n=8). A unilateral ureter obstruction (UUO) model was induced in male rats by ligation of the left ureter as described. Rats were sacrificed at day 14 post-surgery and the obstructed kidneys were harvested and studied. In both groups, the proteinuria, serum creatinine levels were determined, renal morphological changes were observed by HE, masson staining and the location and expression levels of HSP47, collagenlV and fibronectin(FN) were determined using immunohi stochemistry.Results:1. Compared to the sham-operated kidneys, serum creatinine levels increased in the rats of UUO model group, but there was no significant difference in terms of proteinuria levels.2. Compared to the sham-operated kidneys, varying degrees of tubular dilatation or atrophy, infiltration of inflammatory cells in interstitial areas, expansion of interstitial space, and bubble-like degeneration of tubular epithelial cells were observed in the rats of UUO model group under light microscopy.3. The results of Masson staining demonstrated that collagen significantly increased around the renal tubule and in interstitial areas in the rats of UUO model group compared to sham-operated kidneys.4. Compared to the sham-operated kidneys, the results of immunohistochemistry demonstrated that the expression of HSP47 significantly increased in the renal tissue of UUO rats model, especially around the renal tubule and in interstitial areas; The expression of collagenⅣsignificantly increased in the renal tissue of UUO rats model, especially in renal tubular basement membrane and interstitial areas; The expression of FN significantly increased, especially in interstitial areas.Conclusion:The expression of HSP47 increased in the kidneys of UUO rats; The location of expression of HSP47 was also the same as that of expressed collagen. These hint HSP47 could probably promote renal interstitial fibrosis, and the effect was related to promote biosynthesis of collagen. Objective:To study the role of HSP47 in the synthesis of collagen, FN and other ECM proteins by HK-2 cells induced by TGF-β1, and determine its effect on PAI-1 expression, to explore its role in the balance of ECM synthesis and degradation.Method:Human proximal tubular epithelial cells (HK-2) were divided into three groups:control (only culture medium), TGF-β1 (treated with TGF-β1) and HSP47siRNA (treated with TGF-β1 and HSP47siRNA). The expressions of HSP47、collagenⅣ、FN、PAI-1 mRNA and HSP47、collagenⅣ、FN protein were detected by RT-PCR and Western blot respectively.PAI-1 protein was detected by ELISA.Results:1. HK-2 can express HSP47 under normal medium. The expression of HSP47 gene and protein gradually increased with different concentrations of TGF-β1 (0、2.5、5、10ng/ml) intervening with HK-2 at different times(12h、24h、48h), the gene and protein level of HSP47 were highest with 10ng/ml TGF-β1 intervening at 48h on HK-2 cells. 2. Due to the effect of different concentrations of TGF-β1 (0、2.5、5、10ng/ml)at different time points(12h、24h、48h), the levels of gene and protein of collagenⅣ, FN, PAI-1 gradually increased, the gene and protein levels of collagenⅣ, FN, PAI-1 were strongest with TGF-β1 10ng/ml intervening at 48h on HK-2 cells.3. HSP47siRNA Group (HSP47siRNA intervenes at 24h, then TGF-β1 10ng/ml intervenes at 48h) can significantly reduce the expression of HSP47, collagenⅣ, FN, PAI-1 mRNA and protein compared to the intervention only by TGF-β1 on HK-2 cells.Conclusion:l.TGF-β1 can upregulate the expression of HSP47 in a concentration and time dependent mode on HK-2 cell.2. TGF-β1 can upregulate the expression of collagenⅣ, FN, PAI-1 in a concentration and time dependent mode on HK-2 cell.3. HSP47siRNA can reduce the expression of collagenⅣ, FN, PAI-1 mRNA and protein induced by TGF-β1 on HK-2. Objective:To investigate whether mitogen-activated protein kinase (MAPK) channels mediate the up-regulation of expression of HSP47 in HK-2 cells induced by TGF-β1.Methods:MAPK inhibitor was applied to observe the impact of up-regulation of expression of HSP47 in HK-2 cells induced by TGF-β1.Results:PD98059 and SP600125 can specifically inhibit the MAPK pathways:extracellular signal-regulated protein kinase (ERK1/2) and c-Jun-N-terminal kinase (JNK) respectively, and have different effects on the expression of HSP47 up-regulated by TGF-β1.1. PD98059 can inhibit the activation of ERK1/2 signaling pathway and the up-regulation of expression of HSP47 induced by TGF-β1 on HK-2 cells, and it was different among the effect with different concentrations of PD98059 (25uM、50uM、75uM)2. SP600125 can inhibit the activation of JNK signaling pathway and the up-regulation of expression of HSP47 induced by TGF-β1 on HK-2 cells, and there was no difference among the effect with different concentrations of SP600125 (10uM、20uM、30uM)Conclusion:ERK1/2、JNK signaling pathway may be involved in mediating the up-regulation of expression of HSP47 induced by TGF-β1 in HK-2 cells.更多还原