高糖通过转化生长因子依赖途径介导肾小管上皮细胞-肌纤维母细胞转分化及Ⅰ型胶原合成的机制

目的探讨高糖介导肾小管上皮细胞转分化及Ⅰ型胶原（Collagen Ⅰ）合成的分子机制。方法含35mmol／L葡萄糖培养液培养大鼠肾小管上皮细胞（NRK52E细胞）,免疫化学方法检测p-Smad2／3核转位,ELISA检测细胞培养上清中TGF-β1浓度,RT-PCR和Western blot方法分别检测α-SMA、E-eadherin、Collagen Ⅰ mRNA和蛋白的表达。观察TGF-β1中和抗体对高糖上述效应的影响。结果高糖促进NRK52E细胞合成和分泌TGF-β1。TGF-β1中和抗体能抑制高糖介导的p-Smad2／3核转位,下调高糖介导的α-SMA和Collagen Ⅰ蛋白表达,上调E-Cadherin蛋白表达（P均〈0．01）。结论高糖介导的肾小管上皮细胞转分化和细胞外基质Collagen Ⅰ的合成依赖于TGF-β1效应。     

BMP-7过表达抑制大鼠肝星状细胞上皮-间叶转化

目的观察慢病毒介导的BMP-7高表达是否能抑制大鼠HSC-T6细胞发生上皮-间叶转化（epithelial to mesenchymal tran-sition,EMT）,并能拮抗TGF-β1的促EMT作用。方法构建大鼠BMP-7慢病毒表达载体,体外感染HSC-T6细胞株,将成功感染BMP-7重组慢病毒的细胞给予TGF-β1（5 ng/mL）或溶酶体处理。Real-time PCR检测α-SMA、S100A4、E-cadherin mRNA转录水平。Western blotting检测α-SMA、S100A4、E-cadherin、Ⅰ型胶原蛋白表达水平。结果 BMP-7慢病毒感染HSC-T6后,无论TGF-β1存在与否,real-time PCR检测到α-SMA、S100A4 mRNA转录下调,E-cadherin转录上调。Western blotting显示α-SMA、S100A4及Ⅰ型胶原蛋白表达下调,E-cadherin蛋白表达上调。实验组与空病毒对照组比较,差异有统计学意义（P〈0.05）,空病毒对照组与空白组比较,差异无统计学意义。结论 BMP-7能有效拮抗TGF-β1对HSC-T6的促EMT作用,可能成为肝纤维化治疗的新途径。     

瘦素对乙醛诱导肝星状细胞活化过程中Ⅰ型胶原及α-平滑肌肌动蛋白表达的影响

目的:观察瘦素对乙醛诱导肝星状细胞(HSC)活化过程中α-SMA和I型胶原表达的影响,旨在从体外探讨瘦素在酒精性肝病中的可能作用。方法:采用SD大鼠肝脏原位灌流消化的方法获得并原代及传代培养HSC,分乙醛(200μmol/L)处理组,瘦素(100nmol/L)处理组及联合处理组(乙醛200μmol/L加瘦素100nmol/L)。用实时荧光定量PCR方法检测各组细胞TGF-β1、Ⅰ型胶原及α-SMA(α-平滑肌肌动蛋白)mRNA表达水平,Western blot检测各组细胞α-SMA表达量,ELISA法检测培养上清中TGF-β1及Ⅰ型胶原含量。结果:①乙醛处理组中TGF-β1和Ⅰ型胶原基因及蛋白表达量明显高于空白对照组(P<0.05),α-SMA表达量与对照组相比差异无显著性意义(P>0.05);②瘦素处理组TGF-β1、Ⅰ型胶原及α-SMA表达量与对照组相比差异无显著性意义(P>0.05);③联合处理组TGF-β1、Ⅰ型胶原及α-SMA表达量明显高于对照组(P<0.05),但TGF-β1、Ⅰ型胶原表达量与乙醛处理组相比差异无显著性意义(P>0.05)。结论:在乙醛诱导肝星状细胞活化过程中,瘦素能协同上调α-SMA的表达;但对TGF-β1及Ⅰ型胶原的表达无影响,Ⅰ型胶原和α-SMA的表达可能是依赖于不同的调控机制来实现。     

肠上皮细胞来源整合素αVβ6对树突状细胞功能的影响

目的:研究肠上皮细胞(intestinal epithelial cells,IEC)来源的整合素αVβ6对骨髓来源树突状细胞(bone marrow-derived dendritic cells,BMDCs)功能的影响.方法:分离、培养小鼠IEC和DC,IEC经卵清蛋白(ovalbumin,OVA)刺激后,多步离心法制备外泌体(exosome).应用免疫胶体金对外泌体中的整合素αVβ6进行定性,通过免疫磁珠技术,分离获取DC,流式细胞仪检测分离后DC纯度.然后将分离后的DC分5组,分别为空白组、OVA组、外泌体组、外泌体+抗整合素αVβ6抗体组、外泌体+羊抗小鼠IgG组,观察各组对脂多糖(lipopolysaccharide,LPS)刺激的反应.ELISA法检测DC在LPS刺激前后IL-12p70的表达.LPS刺激前活性转化生长因子β1(transforming growth factor-β1,TGF-β1)以及总TGF-β1的表达.结果:OVA组与空白组相比,总TGF-β1表达明显增高(226.636±40.355vs176.947±23.072,P<0.05),但活化TGF-β1表达无明显变化(P>0.05).空白组与外泌体+抗整合素αVβ6抗体组相比,活化TGF-β1表达无明显变化(P>0.05),但总TGF-β1表达明显增高(P<0.05).外泌体组和外泌体+羊抗小鼠IgG组与空白组相比,活化TGF-β1和总TGF-β1表达均明显增高(P<0.05).相比空白对照组,LPS刺激48h后外泌体组和外泌体+羊抗小鼠IgG组IL-12p70表达明显降低(P<0.05),OVA组及外泌体+抗整合素αVβ6抗体组IL-12p70表达无明显差异(P>0.05).结论:IEC来源整合素αVβ6能增加DC活性,以及TGF-β1和总TGF-β1的表达量,并拮抗LPS对BMDC的促成熟作用.     

BMP-7对油酸诱导肾小管上皮细胞转分化的影响

目的观察骨形态发生蛋白（BMP）-7对油酸（OA）诱导的人肾小管上皮细胞转分化的拮抗作用。方法应用体外细胞培养技术培养人肾小管上皮细胞株HK-2。分别用不同浓度BMP-7（0、1、5、10、50ng／ml）和一定浓度的OA（400μmol／L）共刺激体外培养的HK-2 48h,收集细胞,采用ELISA法和RT-PCR法检测转化生长因子（TGF）-β1蛋白、mRNA的水平和表达变化,采用RT-PCR法检测α-平滑肌动蛋白（SMA）mRNA的表达变化。结果BMP-7可以显著减少OA诱导的HK-2中TGF-β1和α—SMA mRNA的表达（P〈0．01）。结论BMP-7可以拮抗OA诱导的肾小管上皮细胞转分化。     

Effects of c-myb antisense RNA on TGF-β1 and α1-I collagen expression in cultured hepatic stellate cells

AIM: To investigate the effects of c-myb antisense RNA on cell proliferation and the expression of c-myb, TGF-β1 and α1-I collagen in cultured hepatic stellate cells (HSC) from rats.METHODS: Recombinant retroviral vector of c-myb antisense gene (pDOR-myb) was constructed, and then transfected into retroviral package cell line PA317 by means of DOTAP.The pseudoviruses produced from the resistant PA317 cells were selected with G418 to infect HSCs isolated from rat livers. The cell proliferation was measured by 3-[4, 5-Dimethylthiazolzyl]-2, 5-diphenyl tetrazo-dium bromide (MTT) method.The expression of c-myb, α1-I collagen and TGF-β1 mRNA, and c-myb protein in HSCs was detected with semi-quantitive reverse transeription-polymerase chain reaction (RT-PCR) and Western-blot respectively.RESULTS: HSCs from rats were isolated successfully with the viability >98%. In the pDOR-myb infected HSCs, the cmyb protein expression, cell proliferation,and α1-I collagen and TGF-β1 mRNA expression were repressed significantly compared with their corresponding control groups (P<0.01).CONCLUSION: c-myb plays a key role in activation and proliferation of HSC. c-myb antisense RNA can inhibit cell proliferation, α1-I collagen and TGF-β1 mRNA expression,suggesting that inhibition of c-myb gene expression might be a potential way for the treatment of liver fibrosis.     

Ca(2+) /calmodulin- dependent protein kinase II mediates transforming growth factor-β-induced hepatic stellate cells proliferation but not in collagen α1(I) production

Aim: Hepatic stellate cells (HSC) are the major players in hepatic fibrosis. As a most potent mitogen, transforming growth factor-β (TGF-β) strongly activates HSC and increases intracellular Ca(2+) concentration. Here, we assessed the potential role of Ca(2+) /calmodulin-dependent protein kinase II (CaMKII), a main downstream effector of the Ca(2+) signal in liver fibrogenesis cascade. Methods: A human immortal HSC cell line, LX-2, and primary rat hepatic stellate cells were used in current study. CaMKII blockage and Akt inhibition were performed by KN-93/CaMKIIα siRNA and LY294002, respectively. HSC proliferation was detected by 5-bromodeoxyuridine incorporation assay. Real-time polymerase chain reaction, western blot and enzyme-linked immunosorbent assay were used to measure mRNA, cellular protein and protein in medium, respectively. Procollagen α1(I) expression was detected by immunocytochemistry. The role of CaMKII on TGF-β/Smad-induced collagen α1(I) expression was determined by (CAGA)(12) -MLP luciferase activity assay. Results: TGF-β dramatically increased CaMKII mRNA, and total and phosphorylated CaMKII expression. KN-93 and CaMKIIα siRNA suppressed TGF-β-mediated HSC proliferation. CaMKII interruption blocked TGF-β-elicited Akt activation. LY294002 arrested HSC proliferation and collagen α1(I) production but had no effect on CaMKII. Furthermore, CaMKII led to increased p21 and p27 expression. KN-93 and CaMKIIα siRNA inhibited TGF-β-induced and basal collagen α1(I) production but had no effect on the activity of (CAGA)(12) -MLP luciferase in response to TGF-β stimulation. Conclusion: CaMKII is a pivotal signal in TGF-β-induced fibrogenic cascades by means of stimulating HSC proliferation, and involved in a basal collagen production. Therefore, CaMKII will be a potentially effective target in the development of therapeutic intervention strategies to attenuate hepatic fibrosis.     

Tetrandrine inhibits activation of rat hepatic stellate cells in vitro via transforming growth factor-β signaling

AIM: To investigate the effect of various concentrations of tetrandrine on activation of quiescent rat hepatic stellate cells (HSCs) and transforming growth factor-β (TGF-β) signaling in vitro.METHODS: HSCs were isolated from rats by in situperfusion of liver and 18% Nycodenz gradient centrifugation, and primarily cultured on uncoated plastic plates for 24 hwith DMEM containing 20% fetal bovine serum (FBS/DMEM) before the culture medium was substituted with 2% FBS/DMEM for another 24 h. Then, the HSCs were cultured in 2% FBS/DMEM with tetrandrine (0.25, 0.5, 1,2 mg/L, respectively). Cell morphological features were observed under an inverted microscope, smooth muscleα-actin (α-SMA) was detected by immunocytochemistry and image analysis system, laminin (LN) and type Ⅲprocollagen (PCⅢ) in supernatants were determined byradioimmunoassay. TGF-β1 mRNA, Smad 7 mRNA and Smad 7 protein were analyzed with RT-PCR and Western blotting, respectively.RESULTS: Tetrandrine at the concentrations of 0.25-2 mg/L prevented morphological transformation of HSC from the quiescent state to the activated one, while α-SMA, LN and PCⅢ expressions were inhibited. As estimated by gray values, the expression of α-SMA in tetrandrine groups (0.25, 0.5, 1, 2 mg/L) was reduced from 21.3% to 42.2%(control: 0.67, tetrandrine groups: 0.82, 0.85, 0.96, or 0.96, respectively, which were statistically different from the control, P＜0.01), and the difference was more significant in tetrandrine at 1 and 2 mg/L. The content of LN in supernatants was significantly decreased in tetrandrine groups to 58.5%, 69.1%, 65.8% or 60.0% that of the control respectively, and that of PCⅢ to 84.6%, 81.5%,75.7% or 80.7% respectively (P＜0.05 vs control), with no significant difference among tetrandrine groups. RTPCR showed that TGF-β1 mRNA expression was reduced by tetrandrine treatments from 56.56% to 87.90% in comparison with the control, while Smad 7 mRNA was increased 1.4-4.8 times. The TGF-β1 mRNA and Smad 7 mRNA expression was in a significant negative correlation (r= -0.755, P＜0.01), and both were significantly correlated with α-SMA protein expression (r = -0.938, P＜0.01;r = 0.938, P＜0.01, respectively). The up-regulation of Smad 7 protein by tetrandrine (1 mg/L)was confirmed by Western blotting as well.CONCLUSION: Tetrandrine has a direct inhibiting effect on the activation of rat HSCs in culture. It up-regulates the expression of Smad 7 which in turn blocks TGF-β1 expression and signaling.     

转换生长因子-β_1对肺成纤维细胞窖蛋白-1与细胞外基质表达的影响

目的 研究转换生长因子-β_1(TGF-β_1)对人胚肺成纤维细胞(HFLF)的窖蛋白-1、Ⅰ型胶原和α-平滑肌肌动蛋白(α-SMA)表达的影响.方法 体外培养HFLF,以未加TGF-β_1的HFLF为对照组.分别以不同终浓度(2、5和10 μg/L)TGF-β_1处理的HFLF为实验组,每组5×10~8细胞,实验重复3次,逆转录-PCR和Western blot法分别检测TGF-β_1刺激3、6、12和24 h后各组窖蛋白-1及其mRNA、Ⅰ型胶原和α-SMA蛋白表达.组间均值比较采用单因素方差分析,采用直线回归模型进行相关性分析.结果 TGF-β_1作用后,HFLF窖蛋白-1的mRNA和蛋白表达逐渐减少,呈时间-浓度依赖关系;TGF-β_1作用12 h,中剂量组(5 μg/L)窖蛋白-1的mRNA和蛋白表达的积分吸光度值(0.59±0.06和0.53±0.04)明显低于对照组(1.22±0.12和1.45±0.06),差异均有统计学意义(F值分别为29.279和95.786,均P＜0.01);TGF-β_1作用24 h,中剂量组(5 μg//L)Ⅰ型胶原和α-SMA蛋白表达的积分吸光度值(1.35±0.09和0.75±0.06)明显高于对照组(0.28±0.04和0.18±0.04),差异均有统计学意义(F值分别为81.221和65.477,均P＜0.01);相关分析结果显示,HFLF窖蛋白-1的蛋白表达下调与Ⅰ型胶原和α-SMA蛋白表达上调旱显著负相关(r值分别为-0.923和-0.793,均P＜0.05).结论 TGF-β_1可下调HFLF窖蛋白-1的mRNA和蛋门表达,并与Ⅰ型胶原和α-SMA蛋白表达上调呈负相关,提示HFLF窖蛋白-1下调可能与肺间质纤维化的发生和发展有关.     

PAP-M6P与RGDY对肝星状细胞活化功能的联合干预作用

背景：活化的肝星状细胞（HSC）表面6-磷酸甘露糖，胰岛素样生长因子Ⅱ（M6P／IGFⅡ）受体和整合素受体显著增加。外源性含有M6P或精氨酰-甘氨酰-天冬氨酸（RGD）基团的分子可干预HSC活化，但目前尚缺乏两者比较或联合应用的研究。目的：探讨人工化学合成的p-氨基苯基-6-磷酸-α-D-甘露糖（PAP—M6P）和精氨酰-甘氨酰-天冬氨酰-酪氨酸（RGDY）小肽对HSC活化功能的联合干预作用。方法：取培养10d的活化期HSC，分别设空白对照组、RGDY对照组、PAP-M6P低、中、高浓度组以及低、中、高浓度联合组进行干预。以甲基噻唑基四唑（MTT）法检测细胞抑制率，以逆转录聚合酶链反应（RT—PCR）检测转化生长因子（TGF）-β1 mRNA表达，以酶联免疫吸附测定（ELISA）检测Ⅰ型胶原、透明质酸和活化的TGF-β1蛋白含量。结果：各组细胞抑制率无明显差异。中浓度联合组TGF-β1 mRNA表达和中、高浓度联合组活化的TGF-β1蛋白含量显著低于RGDY对照组和相应浓度PAP-M6P组（P〈0．05）；高浓度联合组Ⅰ型胶原、透明质酸含量显著低于RGDY对照组和各浓度PAP-M6P组（P〈0．05）。结论：PAP-M6P与RGDY联合应用能显著抑制HSC表达和激活TGF-β1,并减少细胞外基质沉积。     

五灵胶囊调节TGF-β／Smad信号通路蛋白对胶原I、Ⅲ型表达的影响

目的探讨五灵胶囊（WL）调节肝纤维化大鼠肝组织及星状细胞（HSC）TGF-β／Smads信号转导蛋白对胶原I和Ⅲ型（COL～I、COL-Ⅲ）表达的影响。方法SD大鼠以高脂低蛋白饲养、饮用10％乙醇,皮下注射40％CCl4,隔4天注射1次,连续6周制备肝纤维化模型,灌胃给予WL3．86g／kg和秋水仙碱0．1mg／kg治疗1月;另体外分离HSC,以不同剂量的WL与HSC培养24h,最后3h加入TGF-β1 10ng／ml。实验结束后镜检肝细胞变性和肝组织纤维化程度,同时ELISA法测定血清COL-I、COL-Ⅲ和培养液COL—I含量,RT-PCR检测肝组织COL-I、COL-Ⅲ、TGF-β1 mRNA的表达。Western blot检测肝组织及HSC内COL—I、ProCOL—I、LN、α—SMA、ERK、p-ERK、TβRI、TβRⅡ、Smad2／3、Smad7蛋白表达。结果WL组大鼠肝细胞变性和肝纤维化程度明显减轻,血清COL-I、CoL-Ⅲ含量增加,COL-I、COL-ⅢmRNA表达降低。Western blot结果显示,WL能明显下调肝组织α—SMA、LN、p-ERK、TβRI、COL—I、ProCOL-I蛋白表达,对ERK、TβRⅡ、Smad2／3、Smad7蛋白表达无影响;显著下调HSCα—SMA、TβRII、p-ERK、Smad7表达,呈浓度依赖性抑制HSC分泌COL-I。结论WL抑制肝纤维化大鼠肝组织COL-I、COL-Ⅲ合成并增加COL—I降解,其作用位点是下调肝组织和HSCTGF-β／Smad、Ras／ERK信号通路蛋白p—ERK、TβRⅡ表达。     

间接接触共培养下骨髓间充质干细胞分化为心肌样细胞及相关调控基因的时序表达

目的:研究间接接触共培养条件下骨髓间充质干细胞(mesenchymal stem cells,MSCs)向心肌细胞(myocadium-lkce cells,CMs)的分化及相关调控基因的时序表达;筛选MSCs定向分化为CMs的重要调控基因。方法:将MSCs与CMs按1∶5的比例进行间接接触共培养,连续观察两周,在相差显微镜下观察MSCs的形态变化。采用免疫荧光染色法检测心肌特征性肌动蛋白α(α-actin)和心脏肌钙蛋白T(cTnT)的表达。应用半定量RT-PCR分析TGF-β、Nkx-2.5、GATA-4、MEF-2C及TEF-1等相关调控基因在分化过程中的时序表达。结果:共培养后,MSCs的体积增大,其梭形形态逐渐变短变粗近似棒状或椭圆形,细胞之间形成连接,排列方向趋于一致。共培养两周时,α-actin及cTnT阳性细胞的比例分别为29.63%和27.38%。TGF-β、Nkx-2.5、GATA-4和MEF-2C基因在共培养后1 d表达开始增强,诱导后7 d达高峰,以后虽有所下降但仍维持在较高水平;TEF-1基因在诱导过程中表达无明显变化。结论:间接接触共培养条件下,MSCs可分化为心肌细胞。在此过程中,TGF-β、Nkx-2.5、GATA-4和MEF-2C基因可能是调控MSCs定向分化为心肌样细胞的重要调控基因。     

重组转化生长因子-β3基因对大鼠肝星状细胞内、外蛋白表达的影响

目的探讨重组转化生长因子-β3（TGF-β3）基因对大鼠肝星状细胞（HSC）内、外蛋白合成及分泌的影响。方法构建质粒pcDNA3.1（＋）-TGF-β3和pcDNA3.1（＋）-TGF-β1。稳定转染：通过脂质体介导的方法,将pcDNA3.1（＋）-TGF-β1转染HSC-T6,经G418筛选建立稳定高表达pcDNA3.1（＋）-TGF-β1的HSC-T6细胞阳性克隆。再将pcDNA3.1（＋）-TGF-β3转染HSC-T6克隆细胞,用W estern b lot法和酶联免疫吸附法（ELISA）分别检测细胞内外TGF-β1、Ⅰ型胶原、基质金属蛋白酶（MMP）-2、MMP-9及金属蛋白酶组织抑制剂（TIMP）-1的蛋白表达量。结果pcDNA3.1（＋）-TGF-β3转染HSC-T6阳性细胞克隆后,与单纯阳性克隆组相比,TGF-β1、Ⅰ型胶原、TIMP-1细胞内蛋白表达及培养上清中的分泌水平均明显降低（P〈0.05）,MMP-2的表达也降低,但两者间差异无统计学意义（P〉0.05）,而MMP-9的表达明显增高（P〈0.05）。结论重组质粒pcDNA3.1（＋）-TGF-β3能减少细胞内外Ⅰ型胶原的合成及分泌;通过调节MMP-9及TIMP-1的表达,可抑制细胞外基质的合成,促进其降解。     

芪苈强心提取物阻断Smad3信号通路抑制血管紧张素Ⅱ诱导的心脏成纤维细胞转分化

目的观察芪苈强心提取物对血管紧张素II（Angiotensin II,Ang II）诱导的心脏成纤维细胞转分化的影响,探讨芪苈强心胶囊在改善心肌重构方面的分子机制。方法采用胰酶消化法培养新生SD大鼠的心脏成纤维细胞（Cardiac fibroblasts,CFs）,传代后将CFs细胞随机分为：对照组,Ang II（10-6mol/l）组,芪苈强心提取物（0.5mg/ml）组和Ang II＋芪苈强心提取物组。以芪苈强心预处理CFs细胞1h,再以Ang II干预6h,采用免疫荧光和Western Blot等方法分别检测α-SMA,TGF-βl以及TGF-βl下游信号通路中Smad3和Smad6蛋白及磷酸化表达水平。结果与对照组相比,Ang II组CFs的α-SMA、TGF-βl蛋白表达增加。与Ang II组比较,芪苈强心提取物能减少Ang II诱导的α-SMA、TGF-βl蛋白表达,同时降低Smad3蛋白的磷酸化水平,增加Smad6蛋白的表达。结论芪苈强心提取物通过调控TGF-βl/Smads信号通路,实现抑制Ang II诱导的CFs转分化,这可能是芪苈强心改善心肌重构的重要分子机制之一。     

Transforming growth factor-β1 induces intestinal myofibroblast differentiation and modulates their migration

AIM： To investigate the effects of transforming growth factor β1 （TGF-β1） on the differentiation of colonic lamina propria fibroblasts （CLPF） into myofibroblasts in vitro.
METHODS： Primary CLPF cultures were incubated with TGF-β1 and analyzed for production of m-smooth muscle actin （α-SMA）, fibronectin （FN） and FN isoforms. Migration assays were performed in a modified 48-well Boyden chamber. Levels of total and phosphorylated focal adhesion kinase （FAK） in CLPF were analyzed after induction of migration.did not change α-SMA levels, while TGF-β1 treatment for 6 d significantly increased α-SIVlA production. Short term incubation （6 h） with TGF-β1 enhanced CLPF migration, while long term treatment （6 d） of CLPF with TGF-β1 reduced migration to 15%-37% compared to untreated cells. FN and FN isoform mRNA expression were increased after short term incubation with TGF-β1 （2 d） in contrast to long term incubation with TGF-β1 for 6 d. After induction of migration, TGF-β1-preincubated CLPF showed higher amounts of FN and its isoforms and lower levels of total and phosphorylated FAK than untreated cells.
CONCLUSION： Long term incubation of CLPF with TGF-β1 induced differentiation into myofibroblasts with enhanced α-SMA, reduced migratory potential and FAK phosphorylation, and increased FN production. In contrast, short term contact （6 h） of fibroblasts with TGF-β1 induced a dose-dependent increase of cell migration and FAK phosphorylation without induction of α-SMA production.     

Inhibitory effects of microRNA 19b in hepatic stellate cell-mediated fibrogenesis

Hepatic stellate cell (HSC) activation is a pivotal event in initiation and progression of hepatic fibrosis and a major contributor to collagen deposition driven by transforming growth factor beta (TGF-β). MicroRNAs (miRs), small noncoding RNAs modulating messenger RNA (mRNA) and protein expression, have emerged as key regulatory molecules in chronic liver disease. We investigated differentially expressed miRs in quiescent and activated HSCs to identify novel regulators of profibrotic TGF-β signaling. miR microarray analysis was performed on quiescent and activated rat HSCs. Members of the miR-17-92 cluster (19a, 19b, 92a) were significantly down-regulated in activated HSCs. Because miR 19b showed the highest fold-change of the cluster members, activated HSCs were transfected with miR 19b mimic or negative control and TGF-β signaling and HSC activation assessed. miR 19b expression was determined in fibrotic rat and human liver specimens. miR 19b mimic negatively regulated TGF-β signaling components demonstrated by decreased TGF-β receptor II (TGF-βRII) and SMAD3 expression. Computational prediction of miR 19b binding to the 3' untranslated region of TGF-βRII was validated by luciferase reporter assay. Inhibition of TGF-β signaling by miR 19b was confirmed by decreased expression of type I collagen and by blocking TGF-β-induced expression of α1(I) and α2(I) procollagen mRNAs. miR 19b blunted the activated HSC phenotype by morphological assessment and decreased smooth muscle α-actin expression. Additionally, miR 19b expression was markedly diminished in fibrotic rat liver compared with normal liver; similarly, miR 19b expression was markedly down-regulated in fibrotic compared with normal human livers. CONCLUSION: miR 19b is a novel regulator of TGF-β signaling in HSCs, suggesting a potential therapeutic approach for hepatic fibrosis.     

乙酰半胱氨酸抑制人肺成纤维细胞增殖及胶原合成机制的初步探讨

目的 研究乙酰半胱氨酸(NAC)抑制人肺成纤维细胞增殖和胶原合成的机制.方法 分离培养人肺成纤维细胞,并以肺泡上皮来源的细胞系A549作为对照.将细胞分为对照组(不加任何处理)、转化生长因子-β_1(TGF-β_1)组(TGF-β_1为5μg/L)、NAC组(NAC为20 mmol/L)和联合组(5μg/L的TGF-β_1刺激24 h后换用不同浓度NAC继续作用24 h).用不同浓度NAC处理后,四甲基偶氮唑蓝法检测细胞增殖,流式细胞仪检测细胞周期;采用TGF-β_1刺激后加用NAC刺激,逆转录-PCR法检测Ⅰ型前胶原mRNA表达的变化,Western blot法检测cyclin E、α-肌动蛋白及信号转导与转录激活因子-3(STAT-3)蛋白的表达.结果 5、10、20、40 mmol/L的NAC对人肺成纤维细胞生长均有明显抑制作用,呈剂量依赖关系,而20 mmol/L的NAC对A549细胞无明显作用.10、20、40 mml\L的NAC可使G_0/G_1期细胞比例明显升高,S期细胞比例明显降低.经TGF-β_1刺激后,成纤维细胞Ⅰ型前胶原mRNA表达明显增加,而NAC可抑制TGF-β_1诱导或未经诱导的Ⅰ型前胶原表达.TGF--β_1可诱导cyclin E和α-肌动蛋白的表达,其相对密度分别为0.98±0.09和1.56±0.23,20 mmol/L的NAC能明显抑制cyclin E的表达,其相对密度为0.52±0.04,但却小能抑制α-肌动蛋白的表达.TGF-β_1组和NAC组α-肌动蛋白的表达(相对密度分别为1.56 ±0.23和1.63±0.20)无明显差别.结论 NAC能够抑制人肺成纤维细胞cyclin E蛋白的表达,使细胞增殖阻滞于G_1期,从而抑制成纤维细胞发生增殖,抑制其胶原合成.对TGF-β_1刺激前后α-肌动蛋白的表达无明显抑制作用.     

Epithelial responses to lung injury: role of the extracellular matrix

The key role of extracellular matrices in alveolar epithelial cell (AEC) biology is highlighted by the phenotypes of primary AECs cultured on a soft laminin gel contrasted with that on a stiff, fibronectin matrix. On laminin, AECs maintain an epithelial phenotype, and progenitor cells within this population proliferate. In contrast, on fibronectin, AECs rapidly lose surfactant expression and spread extensively, changes that depend on activation of latent TGF-β1 by engagement of fibronectin-binding integrins. The progenitor subpopulation responding to TGF-β1 undergoes epithelial mesenchymal transition (EMT). Although it remains uncertain to what degree EMT contributes directly to collagen 1 production, signaling pathways critical to EMT are important for repair and fibrosis, implying that EMT is part of the general program of lung repair. EMT reprogramming requires not only Smad signaling but also pY654-β-catenin. Generation of pY654-β-catenin requires assembly of complexes of the integrin α3β1, E-cadherin, and TGF-β1 receptors, and such assembly is a function of cell-cell and cell-matrix contacts. Sequestration of α3β1 or E-cadherin in such contacts prevents complex assembly, TGF-β1 induced pY654-β-catenin generation and EMT. Disruption of these contacts is a signal for the cells to initiate repair. Critical remaining questions center around better definition of direct versus indirect effects of EMT on collagen deposition and the nature of AEC progenitors differentiating during fibrogenesis. Elucidation of specific inhibitors of EMT should further test the question of whether the process is important to fibrosis in vivo and a viable therapeutic target.