苦参碱调控Snail2表达水平抑制转化生长因子β1诱导的人腹膜间皮细胞上皮间充质转分化

目的 研究苦参碱对转化生长因子β1(TGF-β1)诱导腹膜间皮细胞上皮间充质转分化(EMT)后转录因子Snail2的影响.方法 采用TGF-β1刺激人腹膜间皮细胞并同时予不同浓度苦参碱干预处理,实验分为空白对照组、TGF-β1(5 ng/ml)诱导组、TGF-β1+0.4 mg/ml苦参碱干预组、TGF-β1+0.6 mg/ml苦参碱干预组、TGF-β1+0.8 mg/ml苦参碱干预组和TGF-β1+1.0 mg/ml苦参碱干预组.实时荧光定量PCR和Western印迹检测Snail2、上皮标志分子E钙黏蛋白(E-cadherin)和间质标志分子α平滑肌肌动蛋白(α-SMA)、纤维连接蛋白(FN)、胶原Ⅲ(ColⅢ)的表达,Western印迹检测Smad2、Smad3和细胞外调节蛋白激酶1/2(ERK1/2)的蛋白磷酸化水平.结果 TGF-β1(5 ng/ml)刺激能上调人腹膜间皮细胞Snail2、α-SMA、FN和ColⅢmRNA和蛋白的表达水平,上调Smad2、Smad3和ERK1/2的蛋白磷酸化水平,下调E-cadherinmRNA和蛋白的表达水平;苦参碱(0.4、0.6、0.8、1.0 mg/ml)干预处理后能下调Snail2和α-SMA、FN和ColⅢmRNA和蛋白的表达水平以及ERK1/2的蛋白磷酸化水平,上调E-cadherin mRNA和蛋白的表达水平.结论 TGF-β1能诱导人腹膜间皮细胞EMT,苦参碱可能通过ERK1/2信号通路下调Snail2的表达水平来抑制TGF-β1诱导的人腹膜间皮细胞EMT.     

高糖诱导足细胞发生上皮-间叶细胞转分化

目的 通过观察高糖是否引起小鼠足细胞发生上皮-间叶细胞转分化现象,探讨糖尿病肾小球损伤的可能机制。 方法 以小鼠永生化足细胞株为研究对象,予不同浓度葡萄糖（12.5、25、50 mmol/L）处理该细胞36 h,并设低糖（5.6 mmol/L）和甘露醇（50 mmol/L）处理组为对照组。采用蛋白免疫印迹和间接免疫荧光染色方法检测α平滑肌肌动蛋白 （α-SMA）、迁连蛋白（FN）、CD2相关蛋白（CD2AP）和Wilms’肿瘤1基因（WT-1）蛋白的表达。 结果 低糖（5.6 mmol/L）和甘露醇（50 mmol/L）处理条件下小鼠足细胞表达WT-1、CD2AP,基本不表达α-SMA和FN;而予不同浓度葡萄糖处理36 h后,足细胞中α-SMA和FN表达水平呈剂量依赖性上调（P < 0.05）。间接免疫荧光染色结果也显示,与低糖组相比,高糖组中 α-SMA阳性的足细胞比例显著增加（P < 0.05）。同时,蛋白免疫印迹结果还表明高糖可呈剂量依赖性方式下调WT-1和CD2AP的表达（P < 0.05）。 结论 在高糖条件下,足细胞发生向间叶细胞表型的转分化可能是引起足细胞功能失调,进而引起糖尿病肾小球损伤发生的作用机制之一。     

Erbin在肾间质纤维化中的表达和作用

目的 探讨Erbin在肾脏间质纤维化中表达量的变化及上调Erbin对转化生长因子β1（TGF-β1）诱导大鼠近端肾小管上皮细胞（NRK52E）转分化的影响。 方法 体内实验采用SD大鼠5/6肾切除法建立肾纤维化动物模型,收集并检测各组血清中Scr、BUN水平;Masson染色观察肾间质纤维化程度;免疫组化及Western印迹检测Erbin的分布与表达。 体外实验采用TGF-β1（10 μg/L）刺激NRK52E细胞72 h建立上皮细胞-间充质转分化（EMT）细胞模型;免疫荧光及Western印迹法检测E钙黏蛋白（E-cadherin）和α平滑肌肌动蛋白（α-SMA）的表达变化;RT-PCR及Western 印迹法检测Erbin的表达变化。用脂质体2000将质粒 Prk5-myc-Erbin瞬时转染至NRK52E细胞,Western印迹法观察上调Erbin表达后对上述各种指标的影响。 结果 （1）假手术组大鼠肾功能正常[Scr（33.96±7.28） μmol/L、BUN（8.11±2.55） mmol/L],Masson染色未见肾间质纤维化,Erbin在肾小管表达较少;模型组大鼠Scr [（140.52±61.11） μmol/L]、BUN[（34.23±7.66） mmol/L] 均显著高于假手术组（均P < 0.05）,肾间质可见明显纤维化,Erbin 在肾小管表达也明显增加,是假手术组的2.9倍（P < 0.01）。（2）正常NRK52E 细胞表达E-cadherin,少量表达Erbin和α-SMA。TGF-β1刺激后,NRK52E细胞E-cadherin表达显著减少,Erbin和α-SMA则表达增加（均P < 0.05）;而转染质粒Prk5-myc-Erbin可逆转TGF-β1诱导的NRK52E细胞E-cadherin表达下调,并可抑制α-SMA表达上调（均P < 0.05）。 结论 Erbin在肾间质纤维化中表达增加,上调Erbin表达可抑制TGF-β1诱导NRK52E发生EMT, 提示Erbin在肾脏纤维化中可发挥保护作用。     

高糖刺激对体外培养小鼠足细胞snail表达和转分化的影响

目的：研究高糖刺激对体外培养的小鼠足细胞snail表达及上皮细胞-间充质细胞转分化的影响.方法：以体外培养的小鼠永生化足细胞为研究对象,将其分为正常糖组、高糖刺激组、甘露醇对照组,培养时间为48 h.倒置显微镜下观察各组足细胞形态,采用荧光定量PCR法检测各组足细胞snail、synaptopodin、α-平滑肌肌动蛋（α-SMA）mRNA的表达,采用免疫细胞化学和western blot检测各组足细胞snail、synaptopodin、α-SMA蛋白的表达量.结果：正常状态下足细胞呈树枝分叉状,几乎不表达snail,高糖刺激48 h后足细胞形态发生明显改变,并且snail、α-SMA mRNA和蛋白表达量较对照组明显上调,synaptopodin表达较对照组减少（P＜0.05）.结论：高糖刺激可诱导足细胞snail的表达和转分化的过程,这可能参与了糖尿病肾病的发生、发展过程.     

Histone methyltransferase MLL1 accelerates the development of renal fibrosis via promoting the epithelial-mesenchymal transition of HK-2 cells

Objective To investigate the possible effects of histone methyltransferase MLL1 on renal interstitial fibrosis and epithelial-mesenchymal transition (EMT). Methods Forty-two male SD rats were randomly divided into normal group, sham operation group and unilateral ureteral occlusion (UUO) group, and then UUO group was further divided into group 1 d, 1 week, 2 week, 3 week and 4 week after operation. The expression of MLL1, E-cadherin, α-SMA, Vimentin and Col3α1 in UUO rat kidney tissue as well as TGF-β1 stimulated HK-2 cells were detected by real-time PCR and Western blotting. siRNA-MLL1 plasmids was used to inhibit the expression of MLL1 and the protein levels of MLL1, α-SMA, Vimentin, E-cadherin, Col3α1 and H3K4me3 induced by TGF-β1 stimulation were detected by Western blotting. The level of H3K4me3 in promoter region of EMT related genes was observed by chromatin immunoprecipitation (CHIP). Results Compared with normal and sham operated groups, the loss of renal function in UUO group was more obvious with the obstruction time (P＜0.05). The renal fibrosis was most obvious 1 week and 2 weeks after the rats underwent the UUO operation (all P＜0.05), with the highest protein expressions of MLL1, E-cadherin, α-SMA, Vimentin and Col3α1 (all P＜0.05). Compared with the control group, 3 ng/ml TGF-β1 induced the highest expression of MLL1 and the most obvious EMT in HK-2 cells (all P＜0.05). Moreover, the EMT and the high level of H3K4me3 in HK-2 triggered by TGF-β1 were all inhibited by siRNA-MLL1 plasmids transfection (all P＜0.05). Conclusions MLL1 can enhance the occurrence of EMT induced by TGF-β1 in HK-2 cells by increasing the level of H3K4me3 in the promoter region of α-SMA and Vimentin.     

Ursolic acid inhibits high glucose-induced epithelial mesenchymal transition of podocyte by mediating Wnt/β-catenin signaling pathway

Objective To observe the epithelial mesenchymal transition (EMT) of podocyte induced by high glucose, and to explore the potential protective mechanism of ursolic acid (UA). Methods The podocytes cultured in vitro were divided into four groups: normal group (glucose 5.5 mmol/L), mannitol group (glucose 5.5 mmol/L+mannitol 19.5 mmol/L), high glucose group (glucose 25 mmol/L) and UA group (glucose 25 mmol/L+UA 5 μmol/L). Podocyte morphology changes were observed by inverted phase contract microscope. The expression of zonula occludens-1 (ZO-1) and α-smooth muscle actin (α-SMA) were detected by immunofluorescence. The expressions of β-catenin and glycogen synthesis kinase-3β (GSK3β) were detected by Western blotting. The expressions of Wnt1, Wnt3a, Wnt5a, Wnt5b and GSK3β were detected by real-time PCR. Results Podocytes showed irregular arborization shape in normal glucose and transited to longer cobblestone-like shape as mesenchyme cell by high glucose culture. Compared with normal group, the expression of ZO-1 protein was down-regulated and the expression of α-SMA was up-regulated by high glucose culture (P＜0.05). The expression of Wnt5a mRNA was down-regulated; β-catenin mRNA and protein were up-regulated (P＜0.05); and GSK3β protein was down-regulated by high glucose culture (P＜0.05). Compared with high glucose group, ursolic acid inhibited podocyte EMT, up-regulated the expression of ZO-1 protein, Wnt5a mRNA, GSK3β (P＜0.05), and down-regulated the expressions of α-SMA protein, β-catenin mRNA and protein (P＜0.05). Conclusion Ursolic acid attenuates high glucose induced epithelial mesenchymal transition of podocyte by inhibiting Wnt/β-catenin signaling pathway.     

CIP4对转化生长因子β1诱导的人肾小管上皮细胞-间充质转分化的影响

目的 观察骨架调节蛋白CIP4（Cdc42 interacting protein 4）对转化生长因子β1（TGF-β1）诱导的人肾小管上皮细胞-间充质转分化（EMT）的影响,并探讨其产生的机制。 方法 10 ?滋g/L TGF-β1刺激72 h诱导人近端肾小管上皮细胞（HK-2细胞）向间充质转分化。Western印迹法检测各组细胞内E-cadherin和α-SMA蛋白的表达。倒置显微镜观察细胞形态的变化。根据GenBank人CIP4的完全cDNA序列,设计1条特异性干扰CIP4表达的RNA片段（CIP4-siRNA）和含野生型CIP4的重组真核表达质粒（pcDNA3.1-hCIP4）,利用lipofactamine 2000将其转染HK-2细胞。Western 印迹法检测对照组、TGF-β1刺激组、CIP4-siRNA转染组、pcDNA3.1-CIP4转染组细胞内CIP4、E-cadherin和α-SMA蛋白的表达,共聚焦显微镜观察 E-cadherin和α-SMA蛋白的分布改变;用PI3K-Akt特异性抑制剂渥曼青霉素（wortmannin） 1 μmol/L干预TGF-β1刺激的HK-2细胞48 h,Western 印迹法检测对照组和干预组CIP4表达的变化。 结果 TGF-β1干预后HK-2细胞E-cadherin蛋白表达显著减少（P ＜ 0.05）,α-SMA蛋白表达显著增多（P ＜ 0.05）,细胞形态由典型的上皮细胞向肌成纤维细胞转变,表明TGF-β1诱导肾小管上皮细胞EMT模型成功。CIP4-siRNA抑制TGF-β1诱导的HK-2细胞表达CIP4后,E-cadherin蛋白表达显著增多（P ＜ 0.05）,α-SMA蛋白表达显著减少（P ＜ 0.05）,部分逆转了上述TGF-β1诱导的肾小管上皮细胞EMT。pcDNA3.1-hCIP4转染使CIP4高表达后,HK-2细胞E-cadherin蛋白表达显著减少（P ＜ 0.05）,α-SMA蛋白表达显著增多（P ＜ 0.05）,诱导了肾小管上皮细胞EMT。用渥曼青霉素干预TGF-β1刺激的HK-2细胞48 h,CIP4可能蛋白表达显著减少（P ＜ 0.05）。 结论 TGF-β1通过PI3K-Akt途径上调CIP4表达,CIP4可能进一步参与TGF-β1诱导的肾小管上皮细胞EMT过程。     

High uric acid induces phenotypic transition of renal tubular cells via PI3K/Akt signaling pathway

Objective To investigate the effect and the mechanism of epithelial-mesenchymal transition (EMT) in renal tubular cells induced by uric acid. Methods Normal rat kidney tubular cell line (NRK-52E) were exposed to different concentrations of uric acid (100, 200, 400, 600, 800 μmol/L UA) for 48 hours to induce EMT. Morphological changes of the NRK-52E cells were examined under an inverted phase contrast microscope. The protein expression of E-cadherin, α-SMA, p-Akt and Akt were detected by Western blotting. The distribution of E-cadherin and α-SMA were detected by immunofluorescence. NRK-52E cells were pretreated by different concentrations of LY294002(0, 2.5, 5, 10, 15 μmol/L), the inhibitor of PI3K/p-Akt signaling pathway, and then processed by uric acid (400 μmol/L) for 48 hours. Western blotting was used to detect the protein expression of p-Akt and Akt. NRK-52E cells were then divided into four groups: normal group (N), uric acid group (UA), LY294002 group (LY), uric acid with LY294002 group (UA+LY). The protein expression of E-cadherin and α-SMA were detected by Western blotting, the distribution of E-cadherin, α-SMA and p-Akt were detected by immunofluorescence. Results There was abundant cellular expression of E-cadherin in unstimulated renal tubular cells whereas its expression was significantly decreased in uric acid-stimulated cells (P＜0.05). In addition, uric acid induced de novo expression of α-SMA in contrast to almost negative staining in untreated cells (P＜0.05). p-Akt were obviously increased in high uric acid group (P＜0.05) and Akt changed not significantly (P＞0.05). NRK-52E cells transformed into elongated fibroblast-like cells from cuboidal clustered epithelial cells. These indicated that uric acid has induced EMT and activated PI3K/p-Akt signaling pathway in NRK-52E cells. However, the above effects of uric acid were abolished when p-Akt was blocked by the PI3K inhibitor (10, 15 μmol/L LY294002), indicated that LY294002 has reversed the trend of EMT. Conclusions High uric acid induces phenotypic transition of renal tubular cells probably via activating PI3K/Akt signaling pathway.     

去甲斑蝥素对TGF-β_1诱导的人近端肾小管细胞上皮间质转分化以及转录因子Snail1表达的影响

目的：探讨去甲斑蝥素对体外人源性肾小管细胞株（HK-2）上皮间质转分化以及转录因子Snail1的影响。方法：常规培养HK-2细胞,分为对照组、TGF-β1组、去甲斑蝥素组（NCTD组）。对照组为无血清DMEM培养,TGF-β1组为TGF-β15ng/ml诱导,NCTD组为不同浓度NCTD（0.5、1.0、2.5μg/ml）与TGF-β1（5ng/ml）共同作用,各组作用时间48h。采用RT-PCR、Western blot分别检测α-SMA、E-cadherin与Snail1表达水平的变化。结果：与对照组相比,TGF-β1组α-SMA mRNA和蛋白表达上调（P〈0.05）,E-cadherin mRNA和蛋白表达下调（P〈0.05）;与TGF-β1组相比,NCTD干预组α-SMA mRNA和蛋白表达下调（P〈0.05）,而E-cadherin mRNA和蛋白表达上调（P〈0.05）,且均呈剂量依赖性。与对照组相比,TGF-β1组Snail1 mRNA和蛋白表达上调（P〈0.05）;与TGF-β1组比较,NCTD干预组Snail1 mRNA和蛋白表达下调（P〈0.05）,具有一定的剂量依赖关系。结论：去甲斑蝥素具有抑制肾小管上皮细胞EMT的作用,该作用可能与下调转录因子Snail1的表达有关。     

热休克蛋白72肽结合区对肾小管上皮间质转分化的影响

目的 探讨热休克蛋白72肽结合区在肾小管上皮间质转分化(EMT)过程中的作用和可能机制.方法 应用质粒转染方法分别诱导热休克蛋白72(HSP72)野生型、肽结合区缺失型(HSP72-△PBD)和肽结合区(PBD)的表达.用转化生长因子β1(TGF-β1)刺激大鼠肾小管上皮细胞(NRK-52E)48 h,Western印迹和免疫荧光染色检测细胞E-钙黏蛋白(cadherin),α-平滑肌肌动蛋白(SMA),HSP72和Smad3/磷酸化(p)-Smad3蛋白表达.结果 TGF-β1(10 μg/L)刺激NRK-52E细胞48 h后上调α-SMA和下调E-cadherin蛋白表达水平.Western印迹及细胞免疫荧光显示,过表达HSP72和PBD能明显减轻TGF-β1诱导的NRK-52E细胞E-cadherin蛋白表达下调和α-SMA蛋白表达上调,而过表达HSP72-△PBD不能改变上述蛋白的表达.此外,过表达HSP72和PBD显著抑制Smad3的磷酸化.结论 HSP72抑制Smad3活化和EMT的发生可能与PBD的功能有关.     

黏着斑激酶在转化生长因子β1诱导的人肾小管上皮细胞转分化中的作用

目的 观察转化生长因子（TGF）β1诱导的正常人近端肾小管上皮细胞（HK-2）转分化（EMT）过程中黏着斑激酶（FAK）的表达及下调FAK的表达后对TGF-β1诱导的HK-2细胞转分化进程的影响。 方法 应用TGF-β1（10 μg/L）刺激HK-2细胞,采用RT-PCR、Western印迹和免疫荧光方法分别检测E钙黏蛋白（E-cadherin）、α平滑肌肌动蛋白（α-SMA）、FAK mRNA和蛋白的表达及磷酸化（p）-FAK（Tyr397）的蛋白表达。应用Lipofectmine2000将FAK siRNA转染HK-2细胞,采用Western印迹观察下调表达FAK对上述指标的影响。 结果 TGF-β1刺激后,HK-2细胞α-SMA蛋白和mRNA水平上调,E-cadherin蛋白和mRNA表达下调。FAK蛋白和mRNA随时间的延长表达逐渐增多,48 h达到高峰。p-FAK（Tyr397）蛋白表达趋势与FAK相同。脂质体转染siRNA后FAK的mRNA和蛋白分别下调了50%和41%,下调表达FAK后可以显著抑制TGF-β1诱导的HK-2细胞α-SMA蛋白的上调表达,逆转 E-cadherin蛋白的下调表达。 结论 在TGF-β1诱导的HK-2细胞转分化进程FAK蛋白表达上调,敲低FAK蛋白表达后可以部分减轻EMT的程度,提示FAK在TGF-β1诱导的肾小管上皮细胞转分化和肾脏纤维化中发挥一定的作用。     

Role and mechanism of Angiotensin Ⅱ-induced ChemR23 in podocyte injury

Objective To observe the expression of ChemR23 induced by Angiotensin Ⅱ (AngⅡ) in podocyte and its role in renal injury. Methods Conditionally immortalized mice podocytes were cultured in vitro. Immunofluorescence was used to observe the sub-cellular location of ChemR23. The expressions of ChemR23, Nephrin and Podocin stimulated by different concentrations of AngⅡ were detected by qRT-PCR and Western blotting. Lentivirus targeting ChemR23 was used. The expressions of Nephrin and Podocin and the phosphorylation state of NF-κB P65 were detected by Western Blot. The inhibitor of NF-κB P65 was added to the cultural medium for 2 h before AngⅡ stimulation. The effect of NF-κB P65 inhibitor on AngⅡ-induced expression of Nephrin and Podocin was detected by Western Blot. Results It is showed that ChemR23 was located in cytosol and membrane. Compared with the normal control, the expression of ChemR23 was significantly increased by AngⅡ in mRNA and protein level, while the expressions of Nephrin and Podocin were decreased (P＜0.05). When using Lentivirus vector to interfere the expression of ChemR23, AngⅡ-repressed expressions of Nephrin and Podocin were restored (P＜0.05). Western Blot showed the level of phosphorylated NF-κB P65 was significantly increased by AngⅡ stimulation (P＜0.05), which could be inhibited by interfering the expression of ChemR23. When adding the NF-κB P65 inhibitor, the low expression of Nephrin and Podocin induced by AngⅡ stimulation was restored (P＜0.05). Conclusions AngⅡ can induce ChemR23 expression, which activates NF-κB P65 signaling pathway, and then inhibits the expressions of Nephrin and Podocin. Targeting ChemR23 is a potential way to alleviate podocyte injury caused by AngⅡ.     

Role of JLP on the epithelial to mesenchymal transition in renal tubular epithelial cells

Objective To observe the effect of JLP on transdifferentiation of human renal proximal tubular epithelial cells (HK-2), and to investigate the role of p38 MAPK signaling pathway in this process. Methods The knock-down plasmids of JLP were constructed. HK-2 cells were randomly divided into four groups: negative control cells (Ctrl-shRNA group), knock-down jlp cells (jlp-shRNA group), negative control cells with FGF-2 treatment (FGF-2 group) and knock-down jlp cells with FGF-2 treatment(jlp-shRNA+FGF-2 group). The expressions of JLP, E-cadherin, TGF-β1, α-SMA, p-p38 MAPK protein were detected by Western blotting．After the induction of FGF-2 for 24 hours, the expressions of α-SMA, COL-I, FN were detected by immunocytochemistry. Results Compared with Ctrl-shRNA group, the expression of JLP protein was significantly down-regulated in FGF-2 group. Compared with FGF-2 group, the expressions of TGF-β1, α-SMA, p-p38 MAPK protein were significantly up-regulated, while E-cadherin protein was significantly down-regulated (P＜0.05). Compared with FGF-2 group, the expressions of α-SMA, COL-I, FN immunostaining increased markedly in jlp-shRNA+FGF-2 group. Conclusion Scaffolding protein JLP is critical in preventing EMT in the course of fibrosis through the inhibition of p-p38 activation in HK-2 cells.     

NPHP1 knockdown induces epithelial-mesenchymal transition in Madin-Darby canine kidney cells

Objective To explore the impacts of NPHP1 knockdown on the phenotype of Madin-Darby canine kidney (MDCK) cells. Methods The expression of NPHP1 in MDCK cells was knockdown by siRNA interference. Cells were divided into normal control group, negative control group and siRNA group. The cellular morphology and migration were observed by light microscope. The mRNA expressions and activities of matrix metalloproteinases 2 and 9 (MMP2 and MMP9) were detected by real time PCR and gelatin zymography. The mRNA and protein expressions of E-cadherin, β-catenin, zonula occluden-1 (ZO-1), ZO-1-associated nucleic acid binding protein (ZONAB) and α-smooth muscle actin (α-SMA) were analyzed by real time PCR, Western blotting and immunocytochemistry. Results Compared with those in normal control group, in siRNA group the mRNA expressions of E-cadherin, β-catenin and ZO-1 decreased, and MMP9, MMP2, α-SMA and ZONAB increased after interfering NPHP1 24 h (all P＜0.05); the protein expressions of E-cadherin, β-catenin and ZO-1 decreased and ZONAB and α-SMA increased after 48 h (all P＜0.05), and MDCK cells became elongated with enhanced migration capacity; siRNA cells had decreased expressions of E-cadherin and β-catenin on the membrane, but increased expression of ZONAB in cytoplasm and nucleoplasm after 72 h, and α-SMA was also observed in some interfered cells. Conclusions NPHP1 knockdown induces epithelial-mesenchymal transition in MDCK cells, and ZO-1/ZONAB signaling pathway was activated. These changes may associate with renal interstitial fibrosis of Nephronophthisis type I.     

肝细胞生长因子下调Smurf2拮抗肾小管上皮细胞-间充质细胞转分化

目的 通过观察肝细胞生长因子( HGF)对正常大鼠肾上皮细胞(NRK-52E)转化生长因子β1( TGF-β1)诱导的Smad泛素化调节因子2(Smurf2)表达的影响,探讨HGF拮抗肾小管上皮细胞-间充质细胞转分化(EMT)的分子机制.方法 以NRK-52E细胞为研究对象,给予TGF-β1(5μg/L)处理0～24 h;或部分细胞经HGF(20 μg/L)预处理30 min后,再予TGF-β1(5μg/L)处理1h或48 h;另外部分细胞予以Smurf2质粒表达载体或Smurf2 siRNA 转染24 h后,再予HGF处理24 h.应用Western印迹及间接免疫荧光染色方法检测Smurf2、SnoN、E钙黏蛋白(E-cadherin)、α平滑肌肌动蛋白(α-SMA)和纤连蛋白(FN)的表达.结果 与对照组相比,TGF-β1处理后迅速上调NRK-52E细胞中Smurf2蛋白表达(P＜0.01);同时显著诱导FN和α-SMA蛋白表达,并下调E-cadherin表达.而予HGF预处理细胞后,可快速抑制TGF-β1诱导的Smurf2表达上调(P＜0.01);并逆转TGF-β1介导的SnoN(P＜0.01)、E-cadherin(P＜0.05)、α-SMA(P＜0.01)和FN(P＜0.01)表达变化.此外,在NRK-52E细胞中过表达Smurf2蛋白可部分抑制HGF诱导的SnoN蛋白上调;而抑制Smurf2表达则可进一步促进HGF诱导的SnoN蛋白表达.结论 在肾小管上皮细胞中,HGF可能通过下调Smurf2表达抑制SnoN蛋白发生泛素-蛋白酶体依赖性降解,进而拮抗TGF-β1介导EMT形成.     

糖肾平通过TGF-β1-Smad2/3-ILK信号通路干预高糖＋LPS诱导足细胞上皮间质转分化的分子机制研究

也页目的：探讨糖肾平对高糖环境下脂多糖（ lipopolysaccharide,LPS）刺激足细胞上皮间质转分化的影响,并探讨其作用机制。方法：以体外培养大鼠肾小球足细胞为研究对象,以高糖（25 mmol/L）、LPS（1μg/mL）刺激足细胞建立模型,分为正常组、高糖组、高糖＋LPS组、厄贝沙坦组、抑制剂组、糖肾平小、中、大剂量组。采用Western blotting及RT-PCR方法检测足细胞中转化生长因子-β1（ transforming growth factor-β1,TGF-β1）、Smad2/3、整合素连接激酶（ integrin-linked kinase, ILK）、CD2相关蛋白（CD2AP）、α-平滑肌肌动蛋白（α-Smooth muscle actin,α-SMA）的表达水平。结果：与正常组比较,高糖组和高糖＋LPS组足细胞TGF-β1、ILK、α-SMA蛋白及其mRNA表达明显增加（P〈0.01）,P-Smad2/3蛋白及Smad2/3 mRNA表达明显增加（P〈0.01）,CD2AP蛋白及其mRNA表达明显减少（P〈0.01）;与高糖＋LPS组比较,厄贝沙坦组足细胞P-Smad2/3、α-SMA蛋白表达明显减少（P〈0.01）,TGF-β1蛋白表达减少（P〈0.05）,TGF-β1,Smad2/3,α-SMAmRNA表达明显减少（P〈0.01）,ILK mRNA表达减少（P〈0.05）,CD2AP蛋白及其mRNA表达明显增加（P〈0.01）;糖肾平大、中、小各剂量组足细胞TGF-β1蛋白表达明显减少（P〈0.01）,糖肾平大剂量组足细胞TGF-β1 mRNA表达减少（P〈0.05）,小、中剂量组表达明显减少（P〈0.01）;糖肾平小、中、大各剂量组足细胞P-Smad2/3蛋白及Smad2/3mRNA表达均明显减少（P〈0.01）;糖肾平小、大剂量组足细胞ILK mRNA表达减少（P〈0.05）,中剂量组表达明显减少（P〈0.01）;糖肾平大剂量组足细胞α-SMA蛋白及其mRNA表达减少（P〈0.05）;糖肾平小、中、大各剂量组足细胞CD2AP蛋白及其mRNA表达均明显增加（P〈0.01）。结论：糖肾平能够降低足细胞TGF-β1、ILK蛋白及mRNA表达,降低P-Smad2/3蛋白及Smad2/3 mRNA表达,升高足细胞标志物CD2AP蛋白及mRNA表达,降低间充质细胞标志物α-SMA蛋白及mRNA表达,通过抑制TGF-β1-Smad2/3-ILK信号通路的激活减少足细胞转分化,保护足细胞,可能是其防治糖尿病肾病的作用机制之一。     

肾间质纤维化中DJ-1抑制抗纤维化因子PTEN的表达

目的 观察肾小管上皮细胞转分化过程中PTEN的表达和分布,并研究上调DJ-1 对 PTEN 表达和分布及 PI3K-Akt 通路活化的影响。 方法 以人肾小管上皮细胞为研究对象,10 μg/L TGF-β1 刺激72 h 诱导人肾小管上皮细胞转分化;Western印迹法检测正常组和 TGF-β1 干预组细胞内 PTEN、E-cadherin 和α-SMA 蛋白表达;RT-PCR 法检测两组细胞内 PTEN mRNA 表达水平。脂质体法介导 pEGFP-N1-DJ-1 或空载体转染人肾小管上皮细胞,倒置荧光显微镜和Western印迹鉴定转染效率后,Western印迹法检测正常组、pEGFP-N1-DJ-1 转染组和空载体转染组细胞内 PTEN 蛋白表达;RT-PCR 法检测各组细胞内 PTEN mRNA 表达。pEGFP-N1-DJ-1 转染前 1 h 用 PI3K 抑制剂 LY294002 预处理,Western印迹检测正常组、pEGFP-N1-DJ-1 转染组和空载体转染组及 LY294002 预处理1 h 后 pEGFP-N1-DJ-1 转染组的 p-Akt 和Akt蛋白的表达。激光共聚焦显微镜下观察正常组、TGF-β1 干预组和 pEGFP-N1-DJ-1 转染组细胞内 PTEN 蛋白的分布。 结果 正常组细胞表达 E-cadherin 和 PTEN,几乎不表达α-SMA;TGF-β1干预组α-SMA 表达显著高于正常组（P < 0.05）,而 E-cadherin表达显著低于正常组（P < 0.05）,PTEN mRNA 和蛋白表达均显著低于正常组（P < 0.05）。pEGFP-N1-DJ-1和空载体转染后,细胞绿色荧光表达均在 80% 以上;pEGFP-N1-DJ-1 转染组细胞内 DJ-1 表达显著高于正常组（P < 0.05）,而 PTEN mRNA 和蛋白表达均显著低于正常组（P < 0.05）;pEGFP-N1-DJ-1 转染组p-Akt 表达显著高于正常组（P < 0.05）,但经 LY294002 干预后与正常组表达基本一致。正常组细胞内PTEN 分布于细胞质和细胞核;TGF-β1 干预组细胞质内 PTEN 几乎完全消失,而细胞核PTEN 略有增加;pEGFP-N1-DJ-1 转染组细胞核表达PTEN,但细胞质几乎无 PTEN 表达,这与 TGF-β1干预组相似。 结论 肾间质纤维化中高表达 DJ-1 可抑制 PTEN 表达,并促进 PI3K-Akt 通路活化。     

多种转移潜能不同的人前列腺癌细胞“上皮细胞间质转化态”特性鉴定及其意义

目的:对多种转移潜能不同的人前列腺癌细胞“上皮细胞间质转化态”(EMT)特性进行鉴定,并从粘附因素和细胞骨架蛋白角度分析其骨转移潜能获得的分子机制。方法:用W estern印迹法鉴定LNCaP及其亚细胞系C4、C4-2和ArCaP亚细胞系IF11、IA8,以及PC-3、Du145等细胞中上皮型钙粘素(E-cadherin)、神经型钙粘素(N-cadherin)和波形纤维蛋白(V im entin)的表达差异情况,并分析其在前列腺癌转移过程中的作用。结果:E-cad-herin在PC-3、LNCaP、C4、C4-2中表达较高,但在Du145、IF11、IA8中表达极低;而V im entin的表达情况恰恰与E-cadherin相反;N-cadherin在IF11、IA8细胞中呈现显著的高表达状态。结论:转移潜能不同的人前列腺癌细胞株之间存在EMT表型的表达差异,其中PC-3、LNCaP、C4、C4-2是未发生EMT改变的细胞,Du145、IF11、IA8却是EMT化的细胞。EMT表型差异蛋白在解释前列腺癌转移机制方面占据着重要地位。