TGF-β1/Smad3信号通路在大鼠肾间质纤维化中的表达和作用及全反式维甲酸的干预

The Expression and Effects of TGF-β1/Smad3 Signaling Pathway in Renal Tubulointerstitial Fibrosis of Rats and Intervention of All-Trans Retinoic Acid

作者: 专业:儿科学 导师:覃远汉 年度:2011 学位:硕士  院校: 广西医科大学

Keywords

TGF-β1/ Smad3 signaling pathway, renal tubulointerstitial fibrosis, expression, effects, all-trans retinoic acid, TGF-β1/Smad3 signaling pathway

        目的通过检测单侧输尿管梗阻(unilateral ureteral obstruction, UUO)所致的肾间质纤维化(renal tubulointerstitial fibrosis, RIF)大鼠肾脏组织转化生长因子-β1(transforming growth factor-β1, TGF-β1)和Smad3的表达,初步探讨TGF-β1/Smad3信号通路在RIF中的作用。方法48只雄性6周龄Wistar大鼠随机分为假手术组和UUO组,每组24只。UUO组大鼠麻醉后,背侧切口行左侧输尿管结扎术,假手术组大鼠同样条件下只探及肾包膜。分别于造模后第7、14、21、28天每组各处死6只大鼠,取其左侧肾脏。HE染色和Masson染色行肾脏病理学检查并计算RIF指数,应用免疫印迹(Western blot)和实时荧光定量-PCR(real-time fluorescent quantitative polymerase chain reaction, RT-PCR)检测肾脏组织Smad3蛋白及其mRNA表达,免疫组织化学法检测肾脏组织TGF-β1、Ⅳ型胶原(collagen-Ⅳ, Col-Ⅳ)和纤维连接蛋白(fibronectin, FN)的蛋白表达。结果1.与假手术组大鼠相比较,UUO组大鼠各时间点:(1)病理学检查显示:弥漫性淋巴细胞、单核细胞浸润,肾小管上皮细胞萎缩,胶原纤维化形成, RIF指数显著增高(P<0.01),梗阻时间越长,RIF指数越高;(2)肾脏组织Smad3蛋白及其mRNA表达均显著增高(P<0.05),梗阻时间越长,表达水平越高;(3)肾脏组织TGF-β1、Col-Ⅳ和FN的蛋白表达均显著增高(P<0.05),梗阻时间越长,表达水平越高。2.相关性分析:(1)UUO组大鼠肾脏组织Smad3、TGF-β1蛋白表达与RIF指数均呈显著正相关( r = 0.520、0.727,P< 0. 01);(2)Col-Ⅳ和FN的蛋白表达与RIF指数均呈显著正相关(r= 0.794、0.786,P<0.01);(3) Smad3蛋白表达与TGF-β1、Col-Ⅳ和FN蛋白表达均呈显著正相关(r= 0.592、0.672、0.508,P<0.01)。结论UUO所致RIF大鼠肾脏组织TGF-β1及Smad3表达均显著增高,梗阻时间越长,表达水平越高,RIF程度越重。TGF-β1/Smad3信号通路可能参与了RIF的发生发展。目的探讨全反式维甲酸(all trans retinoic acid, ATRA)对RIF大鼠肾脏组织TGF-β1/Smad3信号通路的影响,并探讨其作用机制。方法雄性Wistar大鼠72只随机分成3组:假手术组、UUO组、ATRA干预组,每组均为24只。采用UUO方法制备RIF模型,各组于术后第7、14、21、28天分别处死6只大鼠,取其左侧肾脏。肾脏组织HE及Masson染色观察病理改变,计算RIF指数,免疫组织化学法检测肾脏组织TGF-β1、Col-Ⅳ和FN的蛋白表达,Western blot及荧光定量PCR分别检测Smad3蛋白及mRNA表达。结果与UUO组比较,ATRA组大鼠各时间点肾脏组织RIF指数、TGF-β1、Col-Ⅳ和FN蛋白表达均显著降低(P<0.05);肾脏组织Smad3的蛋白及其mRNA表达未见显著降低(P>0.05)。结论ATRA可能通过下调UUO大鼠肾脏组织TGF-β1的表达,减少Col-Ⅳ和FN积聚而起到减轻RIF的作用,但可能并非通过降低TGF-β1/Smad3信号通路中的下游蛋白Smad3表达量而减轻RIF。
    OBJECTIVE To explore the expression of transforming growth factor-β1(TGF-β1) and Smad3 in renal tubulointerstitial fibrosis (RIF) of rats after unilateral ureteral obstruction (UUO) and investigate the effects of TGF-β1/Smad3 signaling pathway in RIF.METHOD Forty-eight Wistar male rats (6-week-old) were randomly assigned into sham operation group and UUO group. The left ureter of rats in UUO group were dissociated to be obstructed after anesthesia . The rats in sham operation group were dissociated left ureter only, and not to be obstructed. Six rats of the two groups were killed respectively at 7, 14, 21, 28 days after surgery, the left kidney were collected. Histological changes of renal tubular interstitium were observed by HE and Masson staining, and the index of RIF was calculated. The expression of protein of TGF-β1,collagen-Ⅳ(Col-Ⅳ), fibronectin (FN) were detected by Immunohistochemistry method. Western blot andreal-time fluorescent quantitative polymerase chain reaction (RT-PCR) were respectively performed to detect the protein and mRNA expression of Smad3.RESULT 1.Compared to the sham operation group, at each same pointof time, (1) Pathologic study showed that, renal tubular epithelial cell atrophy collagen fibrosis and diffuse mononuclear cells, lymphocyte infiltration in UUO, group. The index of RIF in UUO group was increased remarkably (P<0. 01), the longer the obstruction, the higher the RIF index; (2) The expression of protein and mRNA of Smad3 in UUO group were all significantly increased, the longer the obstruction, the higher the expression level; (3) The expression of protein of TGF-β1, Col-Ⅳ, FN in UUO group were all significantly increased, the longer the obstruction, the higher the expression level. 2. Correlation analysis showed that: the expression of protein of Smad3 and TGF-β1 in UUO group was positive correlation with the index of RIF( r = 0.520, 0.727, P< 0. 01), the expression of protein of Col-Ⅳand FN were positive correlations with the index of RIF(r= 0.794, 0.786, P<0.01), and the expression of protein of Smad3 was positive correlation with all the expresssion of TGF-β1, Col-Ⅳ, FN(r= 0.592,0.672, 0.508, P<0.01).CONCLUSIONS The expression of TGF-β1 and Smad3 were all increased in renal tissues of UUO rats, The longer the obstruction, the higher the expression level, the more severe the RIF degree. TGF-β1/Smad3 signaling pathway may involved in The development of RIF. OBJECTIVE To observe the effects of ATRA on the expression of TGF-β1 and Smad3 in the renal tissue of rats with RIF and to investigate the mechanisms of ATRA to TGF-β1/Smad3 signaling pathway in RIF.METHOD Seventy-two Wistar male rats (6-week-old) were randomly assigned into three groups: sham operation group, UUO group and ATRA group. Six rats of the three groups were killed respectively at 7, 14, 21, 28 days after surgery. Histological changes of renal tubular interstitium were observed by HE and Masson staining, and the index of RIF was calculated. The expression of protein of TGF-β1, Col-Ⅳ, FN were detected by Immunohistochemistry method. Western blot and real-time RT-PCR were respectively performed to detect the protein and mRNA expression of Smad3.RESULT Compared to the UUO group, at the same point of time, the index of RIF, the expression of protein of TGF-β1, Col-Ⅳ, FN in ATRA group were all significantly decreased; but the expression of protein and mRNA of Smad3 in ATRA group were all not significantly decreased.CONCLUSIONS ATRA may delay the progress of RIF by reduce the expression of protein of TGF-β1, FN, Col-Ⅳ, but may didn’t by decreasing the expression of protein and mRNA of Smad3 in TGF-β1/Smad3 signaling pathway.
        

TGF-β1/Smad3信号通路在大鼠肾间质纤维化中的表达和作用及全反式维甲酸的干预

一、论文部分4-73
    (一) 英文缩略语表4-6
    (二) 摘要6-13
        1. 中文摘要6-9
        2. 英文摘要9-12
    (三) 前言13-15
    (四) 第一部分 TGF-β1/Smad3信号通路在大鼠肾间质纤维化中的表达和作用15-45
        1. 材料与方法15-28
        2. 结果28-32
        3. 讨论32-36
        4. 参考文献36-39
        5. 附图39-45
    (五) 第二部分全反式维甲酸对肾间质纤维化大鼠肾脏TGF-β145-73
        1. 材料与方法45-47
        2. 结果47-52
        3. 讨论52-56
        4. 参考文献56-59
        5. 附图59-73
二、综述73-83
     参考文献79-83
三、文章发表情况83-84
四、致谢84
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