【作者】 许楚瓯；

【导师】 徐钢；

【作者基本信息】 华中科技大学 ， 肾内科， 2012， 博士

【摘要】 肾间质纤维化是各种慢性肾脏疾病(Chronic kidney disease, CKD)进展到终末期所发生的不可避免的结果。肾小管上皮细胞向间充质细胞转分化(EMT)被认为是其中重要的事件之一,在EMT的过程中,上皮标志物如E-钙粘蛋白(E-cadherin),闭合蛋白(occludin)等表达下降,甚至丢失。E-cadherin主要与β-连环素(β-catenin)一起形成细胞粘附复合体,介导细胞之间的粘附,β-catenin是重要的E-cadherin表达调节分子,其入核后可激活抑制E-cadherin表达相关基因的启动子(如Snail, Slug等)；occludin则为细胞紧密连接的重要组成蛋白,介导细胞之间的紧密连接,维持上皮细胞的极性,其表达可以通过极性蛋白Par6来调节。有研究表明,转化生长因子-betal (TGF-β1)是重要的促纤维化因子,可通过经典的Smad信号通路和其他非Smad信号通路(包括MAPK, PI3K/Akt, ERK, RhoGTPase等)来完成其对肾间质纤维化的调节作用。本文研究的目的蛋白Cdc42相关蛋白(Cdc42-interacting protein, CIP4)是RhoGTPase蛋白家族成员之一,是Cdc42的下游效应蛋白,参与细胞骨架蛋白肌动蛋白(actin)的调节,对维持细胞正常的形态起重要的作用。我们的研究结果表明：无论在大鼠5/6肾切除模型还是小鼠单侧输尿管结扎(Unilateral ureteral obstruction,UUO)的模型中,与假手术组相比较,CIP4的表达均上调,且主要分布在肾小管侧基底膜,细胞之间的连接处,而E-cadherin和occludin的表达则下降。体外实验我们使用TGF-pl诱导的肾小管上皮细胞作为主要研究对象,CIP4的表达同样上升,并伴随有E-cadherin和occludin表达的下调,与体内实验结果一致。在无TGF-β1的刺激下单独高表达CIP4,亦可出现上述相关蛋白的变化。同时我们还观察到,无论是在TGF-β1的诱导下还是CIP4高表达情况下,β-catenin与CIP4均存在相互作用,但是细胞总蛋白中β-catenin的表达无明显变化,在核蛋白中的表达则增加。使用特异性siRNA敲除CIP4后,核蛋白内β-catenin表达减少,同时E-cadherin表达恢复,甚至高于对照组的表达水平。进一步的研究表明,在高表达CIP4的情况下特异性敲除β-catenin, E-cadherin的表达并无明显的变化。另一方面,在TGF-β1诱导和CIP4高表达的两组细胞中,极性蛋白Par6(Polarity protein, Par6)表达无明显的统计学差异,但这两组细胞中均存在CIP4与Par6的相互作用,但是在对照组细胞内是则未观察到该现象。综上所述,我们发现,CIP4可通过促进β-catenin入核调节E-cadherin的表达,同样,CIP4也可能通过Par6参与occludin表达的调节。第一部分肾间质纤维化模型中CIP4的表达与分布目的探讨在SD大鼠5/6肾切除模型以及Balb/c小鼠UUO模型中CIP4的表达量的变化与分布。方法SD大鼠5/6肾切除法建立肾脏纤维化模型,Blab/C小鼠通过结扎单侧输尿管建立肾脏纤维化模型。前者采集各组的血清检测肌酐(Scr)和尿素氮(BUN)值；两组均根据Masson染色结果观察肾脏组织纤维化程度；用免疫组织化学染色观察CIP4在模型组和假手术组的表达和分布。Western blotting检测E-cadheri、occludin、 β-cateni、Par6和α-平滑肌肌动蛋白(α-smooth nuscke actin, α-SMA)蛋白表达变化。结果5/6肾切除模型中,手术组动物Scr, BUN水平高于假手术组；手术组Masson染色显示肾间质明显纤维化,部分肾小球硬化,肾小管萎缩,小管上皮细胞脱落,CIP4表达量上调,主要分布在肾小管上皮细胞侧基底膜细胞连接处。UUO模型中,手术组Masson染色显示肾间质有较多纤维组织形成,部分肾小管扩张,部分肾小管萎缩、上皮细胞脱落。CIP4表达也上调,主要分布在肾小管上皮细胞区域。两种动物模型westen blotting结果均显示,CIP4表达量增加(分别为假手术组的3.91倍和3.64倍)伴随着E-cadherin及occludin表达的下降,α-SMA表达增加(P<0.05),β-catenin和Par6表达量无明显变化。结论CIP4在纤维化的组织中表达明显增加,且主要分布于肾小管上皮细胞,提示CIP4与肾间质纤维化关系密切。第二部分TGF-β1诱导的肾小管上皮细胞转分化中CIP4的表达与分布目的探讨大鼠肾小管上皮细胞(Normal rat kidney52E, NRK52E细胞)在TGF-β1诱导下CIP4蛋白表达量的变化。方法NRK52E细胞在含有TGF-β1(10ng/ml)的培养液中培育72小时,光学电子显微镜观察细胞形态的变化；提取细胞蛋白,Western blotting检测CIP4、E-cadherin、 occludin、β-catenin、Par6以及α-SMA蛋白表达的变化。结果对照组细胞呈铺路石样外观,细胞间连接紧密,细胞形态规则；模型组细胞体积变大,形状不规则,细胞间连接松散,间隙变大。Western blotting结果显示与对照组相比,TGF-β1刺激组的NRK52E细胞CIP4表达上调,为对照组的1.72倍,同时α-SMA蛋白表达也增高,E-cadherin及occludin表达量减少(P<0.05),β-catenin和Par6表达量无统计学差异。结论体外实验与体内模型所得到的结果一致,提示CIP4参与TGF-β1诱导的肾小管上皮细胞转分化进程。第三部分CIP4对E-cadherin表达的影响与调节目的探讨在肾小管上皮细胞转分化的过程中,CIP4对P-catenin核转位的调控,以及对E-cadherin表达的影响。方法用脂质体2000将pcDNA4.0/CIP4质粒瞬时转染至NRK52E细胞内,Western blotting检测相关各蛋白的表达；免疫荧光法检测CIP4及β-catenin在细胞内的共定位,用共聚焦显微镜观察；免疫沉淀法检测CIP4与β-catenin的相互作用；用特异性siRNA敲除相关蛋白的表达。结果在TGF-β1刺激组中,CIP4和β-catenin在核蛋白中的表达均增加,分别为对照组的1.82倍和1.59倍。在共聚焦显微镜下观察,免疫荧光结果显示正常组CIP4与β-catenin主要分布于细胞膜,且在细胞膜上存在部分共定位,模型组中,CIP4与β-catenin在细胞膜上分布减少,细胞核内表达增多,细胞核内存在部分共定位现象；同时用免疫沉淀法检测,在这两组细胞中CIP4和β-catenin均存在着相互作用。在倒置显微镜下观察到,对照组细胞形态规则,呈铺路石样的外观,细胞间连接紧密；转染了CIP4质粒的细胞细胞形态不规则,连接松散,体积变大,其改变与TGF-β1刺激后的细胞相类似；而转染了pcDNA4.0空载体的细胞则未发生上述改变。单独转染CIP4目的基因质粒的NRK52E细胞中,CIP4与α-SMA表达上调,E-cadherin表达减少(P<0.05),β-catenin的表达无明显变化。用特异性的siRNA沉默CIP4的表达后,CIP4的表达量只为原来40%。随着CIP4表达的下降,E-cadherin表达升高,甚至高于对照组(P<0.05),同时β-catenin的表达仍无明显的变化。NRK52E细胞转染CIP4质粒后,核蛋白中CIP4与β-catenin表达均增加(P<0.05),特异性敲除CIP4后,CIP4在核蛋白中表达下降,β-catenin的表达也下调(P<0.05)。将β-catenin特异性siRNA转染至NRK52E细胞,β-catenin的表达量下降为对照组的65%,E-cadherin与CIP4表达量无明显改变,在已转染高表达CIP4质粒的细胞中再转染特异性的P-catenin siRNA,与对照组相比,只转染CIP4质粒组的细胞,E-cadherin表达随CIP4表达的增高而下降(P<0.05),而在既转染了CIP4质粒也转染了β-catenin siRNA的细胞中,E-cadherin的表达恢复至对照组水平(P<0.05)。结论CIP4可通过促进β-catenin入核,从而调节E-cadherin的表达。第四部分CIP4对occludin表达的影响目的探讨NRK52E细胞高表达CIP4后对紧密连接蛋白occludin表达的影响及与极性蛋白Par6之间的相互作用关系。方法用脂质体2000将pcDNA4.0/CIP4质粒瞬时转染至NRK52E细胞中,Western blotting检测相关各指标的变化；用免疫沉淀法检测CIP4与Par6之间的相互作用。结果单独高表达CIP4, occluidn表达下降,α-SMA蛋白表达量上升,Par6表达量无明显变化,与TGF-β1作用后相关蛋白改变一致。在TGF-β1刺激组与CIP4质粒转染组的细胞中,存在CIP4与Par6的相互作用。而在对照组细胞中则未观察到该现象。结论CIP4可能通过与Par6相互作用,参与occludin表达的调节。更多还原

【Abstract】 Renal interstitial fibrosis is an inevitable outcome of almost all kinds of chronic kidney diseases (CKD). Epithelial-mesenchymal transition (EMT) is considered as one of the most important-events leading to fibrosis. During the process of EMT, the biomarkers of epithelia, such as E-cadherin and occludin, the expression of which is lost. E-cadherin, form the ’adhesive complex’ with β-catenin together, which is crucial to the cell-cell adhesion. β-catenin is a good candidate to regulate the expression of E-cadherin, β-catenin translocation to the nucleus, then activate the promoter of Snail gene family, the repressor of E-cadhein. Occludin is a component of tight junction, which maintains the polarity of epithelia, is regulated by the polarity protein Par6. Evidences show that transforming growth factor-betal (TGF-β1) is an important cytokine during fibrogenesis, to regulate its function, TGF-β1utilizes multiple signaling pathways by canonical TGF-β/Smad pathway components’cross talks’with other signaling pathways, namely non-Smad pathways, these include various branches of MAP kinase pathways, phosphatidylinositol-3-kinase (PI3K)/AKT pathways, ERK pathways and Rho-like small GTPase signaling pathways. Cdc42-interacting protein4(CIP4), a member of RhoGTPase family, is a downstream effector of Cdc42, and participates the modulation of cytoskeletal protein actin. CIP4is crucial to maintain the normal morphology of cell.In our study, we demonstrated that in both animal models,5/6nephrectomized rats and unilateral unreteral obstructed mouse, CIP4expression was upregulated comparing with sham operated group, and was distributed in renal tubule-interstitial area, mainly at the baso-lateral sides of epithelia, the exact location of cell-cell junctions. CIP4upregulation was companied by downregulation of E-cadherin and occludin. We treated NRK52E cells with TGF-β1to set up a EMT model in vitro, the expression of CIP4was found increased accompaning with the decreased expression of E-cadherin and occludin, which was consistent with the results of in vivo experiments. Over-expression of CIP4without the stimulation of TGF-β1, the related proteins above were show the similar change in protein expression. Also we found, in both TGF-β1treated cells and CIP4over-expression cells, CIP4interacted with β-catenin, the expression of β-catenin had no difference among those groups in the whole cell lystaes. However, the expression of β-catenin in nuclear protein had been elevated in TGF-β1treated and CIP4over-expression cell groups, which was reduced when CIP4was silenced by the specific siRNA, meanwhile, E-cadherin was upregulated in CIP4siRNA-transfected cells. Furthermore, β-catenin knockdown by speeific siRNA while CIP4was over-expressed, E-cadherm expression had no change. On the other hand, in both TGF-β1treated cells and CIP4over-expressed cells, Par6expression had no differences comparing with control group, however, the interaction between CIP4and Par6was observed in these two groups, but not in control group. All above, we conclude that CIP4may repress the expression of E-cadherin by promoting β-catenin translocate to the nucleus, also, CIP4may regulate the expression of occluding by interacting with Par6. Part1The expression and distribution of CIP4in renal fibrotic modelsObjective To investigate the distribution and expression change of CIP4in5/6nephrectomized SD rats and unilateral ureteral obstructive (UUO) Balb/c mouse.Methods The renal fibrotic animal models were induced by5/6subtotal nephrectomy in SD rats and left ureteral obstruction in Balb/c mouse. The serum was collected in SD rats to detect the change of serum creatine (Scr) and blood urea nitrogen (BUN). In both models, the severity of renal fibrosis was detected by Masson staining. Immunohistochemical staining was used to detected the expression and distribution of CIP4. The protein expression of CIP4, β-catenin, E-cadherin. Occludin, Par6and α-SMA were defected by western blotting.Results In the model of5/6nephrectomized rats, the value of Scr and BUN were higher than those in sham operated rats(P<0.05). Masson staining showed sclerotic glomorulus, renal tubular atrophy and epithelial drop. CIP4expression was upregulated, and was mainly distributed in basolateral side of epithlia. In UUO model, Masson stainning also showed sclerotic glomorulus, interstitial fibrosis. Renal tubules were partial enlarged, partial atrophy. Western blotting of both models showed that CIP4was upregulated(3.91-fold and3.64-fold respectively) accompanied by upregulation of α-SMA and downregulation of E-cadherin and occludin (P<0.05), β-catenin and Par6showed no change in protein expression.Conclusions CIP4was found increased in protein expression in fibrotic renal tissue, and was mainly distributed in renal tubular epithelia, which indicated that CIP4was closely related with renal tissue fibrogenesis.Part2The expression and distribution of CIP4in TGF-β1induced renal tubular EMTObjectives To investigate the expression change of CIP4in murine epithelial cell line NRK52E while incubating with TGF-β1.Methods NRK52E cells were incubated with TGF-β1(10ng/ml) for72h, the morphological change was observed by light microscope. The protein expression of CIP4, β-catenin, E-cadherin, occludin, Par6and a-SMA were detected by western blotting.Results The morphological appearance of control cells were showed as cobble-stone in shape, cell-cell junction was tight. After TGF-β1treatment, cells had a large and irregular shape, cell junctions were loosen. Western blotting showed that in TGF-β1treated cells, CIP4was upregulated1.72-fold comparing with control group, together with α-SMA, reduced expression of E-cadherin and occludin were also abserved (P<0.05). The expression of β-catenin and Par6had no difference.Conclusions The results in vitro were consistence with those in vivo, which indicated that CIP4participate in the process of TGF-β1induced EMT in NRK52E cells.Part3Effect and regulation of CIP4to the expression of E-cadherinObjectives To investigate the effect of CIP4to P-catenin translocation to the nucleus, and the expression of E-cadherin.Methods The plasmid pcDNA4.0/CIP4was transient transfected into NRK52E cells by lipofectamine2000, all procedures were followed by the instructions. The colocalization of CIP4and β-catenin was detected by immunofluoresence using confocal microscope. The interaction between CIP4and β-catennin was detected by immunoprecipitation.. CIP4and β-catenin were silenced by specific siRNA respectively.Results The expression of CIP4and β-catenin were elevated in nuclear protein after TGF-β1treatment,1.82-fold and1.59-fold respectively. Under the cofocal microscope, immunofluroscence showed partial colocalization of CIP4and β-catenin at the cell membrane in control cells, the expression of both proteins were elevated and had partial colocalization in the nucleus after TGF-β1incubation. CIP4interacted with β-catenin in both groups detected by immunoprecipitation. In CIP4-transfected NRK52E cells, α-SMA was upregulated accompanied with CIP4, E-cadherin was downregulated (P<0.05), the results was similar as those in cells treated by TGF-β1only. To analyze the nuclear proteins, β-catenin expression was upregulated in CIP4over-expressed cells, E-cadherin expression was reduced in whole cell lysates\(P<0.05). After CIP4was knockdown by the specific siRNA, we observed reduced β-catenin expression in nuclear proteins, and E-cadherin expression was restored. Furthermore, after CIP4over-expression, β-catenin was silenced by siRNA, the E-cadherin expression had no difference.Conclusions CIP4may repress the expression of E-cadherin by promoting translocation of β-catenin to the nucleus. Part4Effect of CIP4on the expression of occludinObjectives To investigate the effect of CIP4over-expression on the expression of tight junction protein occludin, and the interaction between CIP4and Par6in NRK52E cells.Methods The plasmid pcDNA4.0/CIP4was transient transfected by lipofectamine2000into NRK52E cells, all the procedures were followed by the instructions. The protein expression of CIP4, Par6, α-SMA and occludin was detected by western blotting. The interaction between CIP4and Par6was detected by immunoprecipitation.Results CIP4over-expression alone without the stimulation of TGF-β1, increased expression of α-SMA and reduced expression of occludin was observed, which was consistent with the results in TGF-β1induced cells. The interaction between CIP4and Par6was detected in TGF-β1treated cells and CIP4over-expression cells, but t not in control cells.Conclusions CIP4may participate the modulation of expression occludin by interacting with Par6.更多还原