TGF-β1/TGF-βRⅡ和Smad4在葡萄膜黑色素瘤中的表达

目的探讨转化生长因子β1(TGF—β1)／转化生长因子β受体Ⅱ(TGF—βRⅡ)和Smad4在葡萄膜黑色素瘤中的表达以及它们在葡萄膜黑色素瘤发病过程中可能的机制。方法应用免疫组织化学SP法检测TGF—β1／TGF-βRⅡ和Smad4在24例葡萄膜黑色素瘤瘤组织石蜡切片中的表达，并累计其表达的阳性率。结果24例葡萄膜黑色素瘤组织切片中，可见瘤组织TGF-β1阳性率为24／24，TGF-βRⅡ阳性率12／24，Smad4阳性率11／24，后两者双阳性率为5／24。结论葡萄膜黑色素瘤组织中TGF—β1表达高于瘤旁组织；TGF-βRⅡ和(或)Smad4的表达异常与葡萄膜黑色素瘤发病有关。     

Involvement of Rho-associated coiled-coil kinase signaling inhibition in TGF-β1/Smad2, 3 signal transduction in vitro

AIM: To research the effect of Y-27632, a selective Rho-associated coiled-coil kinase (ROCK) inhibitor, on TGF-β1/Smad2, 3 signal transduction in ocular Tenon’s capsule fibroblasts (OTFs). METHODS: Primary ocular Tenon’s capsule fibroblasts had been cultured in vitro. The effect of Y27632 on proliferation of OTF stimulated by lysophosphatidic acid (LPA) was evaluated by MTT colorimetric assay so as to sift out the proper concentrations range of Y-27632 for the next experiment. Real time-polymerase chain reactor (RT-PCR) was to analyze the changes of Smad2 and Smad3 genes of cells affected by Y-27632, though unaffected by transforming growth factor-beta1 (TGF-β1). Proteins of Smad2, Smad3, phosphorylated Smad2 (Ser245/250/255), and phosphorylated Smad3 (Ser423/425/203) were respectively quantified by Western blot after OTFs were successively incubated by TGF-β1 and Y-27632. Meanwhile, α-smooth muscular actin (α-SMA) protein was also quantified after the small intervening gene fragments of human Smad2 and Smad3 were designed, synthesized, and then transfected to OTFs. RESULTS: Y-27632 significantly inhibited OTFs proliferation stimulated by LPA. Also Y-27632 significantly suppressed the expressions of Smad2 mRNA, Smad2, 3 proteins expressions, Smad3 phosphorylation at the carboxylic terminals of Ser423/425/203 which had been radically promoted by TGF-β1. SiRNA-Smad2, 3 suppressed α-SMA expressions, but less effectively than Y-27632. CONCLUSION: The inhibition of ROCK signaling may be a potential therapeutic candidate for the treatment of the filtration channel fibrosis.     

TGF-β1、TGF-βRⅡ及Smad4蛋白在原发性翼状胬肉中的表达

目的研究转化生长因子-β1(transforming growth factor-β1,TGF-β1)、转化生长因子-β1Ⅱ型受体(transforming growthfactor-β1receptorⅡ,TGF-βRⅡ)和Smad4蛋白在原发性翼状胬肉组织中的表达及其意义。方法应用免疫组织化学SP法检测56眼翼状胬肉头部组织及8眼正常结膜组织中TGF-β1、TGF-βRⅡ及Smad4蛋白的表达,阳性表达率用阳性细胞占细胞总数百分率的平均值表示,利用统计学软件SPSS13.0分析本研究结果。结果TGF-β1在胬肉头部侵入角膜缘≤1mm、2~3mm和≥4mm翼状胬肉组和正常结膜组的阳性表达率利用分别为(21.08±3.63)%、(20.12±2.68)%、(21.55±3.07)%、(12.36±1.97)%;TGF-βRⅡ的表达率分别为(15.35±9.37)%、(21.07±10.96)%、(15.48±8.80)%、(33.77±9.31)%,Smad4蛋白的表达率分别为(14.38±4.02)%、(13.05±3.50)%、(13.13±4.10)%、(38.69±5.70)%,TGF-β1在各翼状胬...     

TGF-β/Smad信号传导通路在后囊膜混浊中用机制的研究进展

后囊膜混浊是白内障术后引起视力下降的常见并发症.转化生长因子-β(transforming growth factor-β,TGF-β)是调节晶状体上皮细胞转分化和产生细胞外基质的重要因子.TGF-β主要通过Smad信号传导通路实现其生物学作用,介导晶状体上皮细胞的增生、上皮细胞间充质转化和细胞外基质的产生.本文对TGF-β/Smad信号传导通路及其在后囊膜混浊形成中作用机制的研究现状进行综述.     

Changes of TGF-β2, MMP-2, and TIMP-2 levels in the vitreous of patients with high myopia

Purpose

To investigate the concentrations of transforming growth factor (TGF)-β2, matrix metalloproteinase (MMP)-2, and tissue inhibitor of metalloproteinase (TIMP)-2 in the vitreous of patients with high myopia.

Methods

Twenty-six patients with high myopia (HM) who received vitrectomy for macular retinoschisis or macular hole were enrolled in this prospective study. Twenty-six patients with idiopathic macular hole or macular epiretinal membrane were chosen as a control group. Vitreous samples were obtained during the vitrectomy surgery. The levels of TGF-β2、MMP-2、TIMP-2 in the vitreous samples were measured by enzyme-linked immunosorbent assay. The MMP activity was determined by a fluorometric assay.

Results

There was no significant difference in the vitreous level of TGF-β2 between HM (1.64?±?0.38 ng/ml) and the control group (1.56?±?0.32 ng/ml, p?=?0.56). The vitreous levels of MMP-2 in HM (32.40?±?14.90 ng/ml) were significantly higher than in the control group (21.42?±?6.74 ng/ml, p?MMP-2/TIMP-2 was significantly elevated in the vitreous samples from HM (0.61?±?0.19), compared to the control group (0.48?±?0.11, p?p?Conclusions The elevated MMP/TIMP ratio and MMP activity may play a role in the pathogenesis of human high myopia. Large prospective studies are needed to further investigate the effect of MMPs in the pathogenesis of human high myopia.     

Smad 7抑制TGF-β2的抗角膜内皮细胞增殖效应并加速内皮损伤

随着内眼手术的大量开展 ,角膜内皮的损伤逐渐增多 ,但由于角膜内皮细胞的不可再生性 ,给临床治疗带来许多困难。探索影响内皮修复的因素 ,将成为眼科界的一个课题。研究表明 ,房水中存在着大量的转化生长因子 β2 (TGF β2 ) ,它能上调角膜内皮细胞p2 7kip1 (一种细胞周期蛋白依赖性激酶抑制剂 )的表达 ,而 p2 7kip1的表达抑制角膜内皮细胞的分裂 ,所以TGF β2对角膜内皮细胞增生修复起抑制作用。Smad是一个新的蛋白家族 ,角膜内皮细胞可以表达Smad2、Smad3、Smad4和Smad7。本实验利用Smad7阻断TGF β2在细胞内的信号传导 ,从而拮抗…     

TGF-β2作用于人晶状体上皮细胞后Smad 4与p-Smad2／3的动态表达

目的观察转化生长因子β2（TGF-β2）作用于人晶状体上皮细胞（LECs）后2类通路转导蛋白Smad4和p-Smad2／3的动态表达变化。方法体外培养的人LECs中添加10ng／mL人TGF—β2,RT-PCR方法测定不同时间点Smadg mRNA的表达,Western blot观察p-Smad2／3的表达,免疫组织化学法观察p-Smad2／3在LECs表达的特点。结果人LECs添加了外源性人TGF-β2,后1h,可出现Smad 4 mRNA表达增高,16h时达峰值（0．72±0．07）,并持续至24h。与对照组比较差异无统计学意义（P〈0．05）。p-Smad2／3在2h后逐渐增强,与对照组比较差异明显,16h达到峰值（2．53g／L）。16h有p-Smad2／3颗粒胞浆表达,24b向核内聚集。结论TGF-β2刺激LECs后,可激活膜受体激活型Smad和通用型Smad,TGF-β2对Smad通路蛋白的激活存在时效关系。TGF—β2可通过特异性激活Smad通路,将信号转导至细胞核内。     

TGF-β1/Smad4信号传导系统在视网膜母细胞瘤株Hxo-Rb44的表达

目的探讨TGF-β1／Smad4信号传导系统在视网膜母细胞瘤株Hxo-RM4的表达及意义。方法用免疫荧光法检测TGF-β1、TGF-β受体Ⅱ及Smad4在该细胞株中的表达和定位。以已知TGF-β1、TGF-β受体Ⅱ及Smad4表达均为阳性的WI-38细胞作对照，分别用RT-PCR法和Western blot法检测TGF-β1、TGF-β受体Ⅱ及Smad4的mRNA和蛋白质在视网膜母细胞瘤细胞株Hxo-RM4中的表达，并比较两种细胞表达水平的差异。结果免疫荧光检测结果发现视网膜母细胞瘤细胞株Hxo-RM4的TGF-β1呈阳性表达，主要位于胞浆和胞膜；TGF-βRⅡ及Smad4阴性。视网膜母细胞瘤细胞株Hxo-Rbd4的TGF-β1的表达在mRNA水平和蛋白水平均高于作对照的WI-38细胞；TGF-β受体Ⅱ及Smad4在mRNA水平和蛋白水平均为阴性。结论视网膜母细胞瘤细胞株Hxo-Rbd4高表达TGF-β1；对TGF-β1增殖抑制作用反应的丧失与视网膜母细胞瘤的发病有关。     

TGF-β1及Smad4在氧致小鼠视网膜病变中的表达

目的 利用氧致小鼠视网膜病变的模型,研究TGF-β1及其信号传导分子Smad4-mRNA在视网膜上的表达,探讨它们与新生血管形成的关系.方法 实验组出生7 d的小鼠与母鼠一起在75%的氧环境下饲养5 d后再在正常环境下生活5 d;对照组在正常环境下生活.第17d时取两组小鼠的眼球,切片后行苏木精-伊红染色、TGF-β1免疫组织化学及Smad4 mRNA原位杂交检测.结果 给氧组苏木精-伊红染色每个切面突破视网膜内界膜的平均内皮细胞核数目为(22±3.5)个,对照组＜1个.给氧组TGF-β1免疫组织化学及Smad4-mRNA原位杂交检测在视网膜内界膜附近均见较强的阳性颗粒,与内皮细胞增生的部位相一致,对照组的阳性颗粒较少,经统计学分析两组的差别均有显著统计学意义.结论 TGF-β1及Smad4基因在氧致小鼠视网膜病变中的表达显著增加,TGF-β1可能通过Smad途径参与新生血管的形成,对TGF-β途径的研究可能为眼部新生血管疾病的防治提供新的策略.     

Relationship of MMP-2 and TGF-β2 levels in human vitreous with axial length

AIM: To investigate the relationship of matrix metalloproteinase-2 (MMP-2) and transforming growth factor-beta 2 (TGF-β2) levels in human vitreous with axial length (AL) of patients with high myopia. METHODS: The concentrations of MMP-2 and TGF-β2 levels were tested by enzyme-linked immunosorbnent assay (ELISA). Fifty-five human vitreous samples of 55 patients were collected during vitrectomy surgery, and were divided into two groups according to their spherical equivalent (SE) and AL. High myopia group (25 cases): AL≥26.00 mm, and control group or non-high myopia group (30 cases): AL<26.00 mm. RESULTS: The MMP-2 levels in vitreous of high myopia group (96.87±55.95 ng/mL) was significantly higher than that of control group (77.24±41.81 ng/mL, P<0.05), but not correlated with AL (r=0.088, P=0.544). While the vitreous TGF-β2 concentration was negatively correlated with AL (r=-0.344, P=0.014), and there was significant difference of TGF-β2 vitreous levels between high myopia group (3729.08±1890.88 pg/mL) and control group (3926.00± 1333.88 pg/mL, P<0.05). CONCLUSION: MMP-2 and TGF-β2 in human vitreous may play a critical role in human high myopia development, and the TGF-β2 appears to be associated with AL.     

TGF-β1及Smad4在氧致小鼠视网膜病变中的表达

目的利用氧致小鼠视网膜病变的模型,研究TGF-β1及其信号传导分子Smad4-mRNA在视网膜上的表达,探讨它们与新生血管形成的关系。方法实验组:出生7d的小鼠与母鼠一起在75%的氧环境下饲养5d后再在正常环境下生活5d;对照组在正常环境下生活。第17d时取两组小鼠的眼球,切片后行苏木精-伊红染色、TGF-β1免疫组织化学及Smad4mRNA原位杂交检测。结果给氧组苏木精-伊红染色每个切面突破视网膜内界膜的平均内皮细胞核数目为(22±3·5)个,对照组<1个。给氧组TGF-β1免疫组织化学及Smad4-mRNA原位杂交检测在视网膜内界膜附近均见较强的阳性颗粒,与内皮细胞增生的部位相一致,对照组的阳性颗粒较少,经统计学分析两组的差别均有显著统计学意义。结论TGF-β1及Smad4基因在氧致小鼠视网膜病变中的表达显著增加,TGF-β1可能通过Smad途径参与新生血管的形成,对TGF-β途径的研究可能为眼部新生血管疾病的防治提供新的策略。     

翼状胬肉中MMP-2,TIMP-2和TGF-β1的表达意义

目的:探讨MMP-2及其抑制剂TIMP-2和影响它们作用的TGF-β1在翼状胬肉中的表达及意义。方法:采用Maxvision免疫组化技术检测在20例翼状胬肉和10例正常球结膜中MMP-2、TIMP-2及TGF-β1的表达,并比较两组中表达的差异。结果:MMP-2和TGF-β1在翼状胬肉中的表达的阳性率显著高于正常球结膜中它们的阳性表达率(z=-4.618,-4.376,P<0.01),而TIMP-2在两组中的表达差异无显著性(z=-1.319,P>0.05)。经Spearman等级相关检验分析,在翼状胬肉中,MMP-2和TGF-β1的阳性表达率存在正相关(r=0·686,P<0.01);而MMP-2和TGF-β1与TIMP-2阳性表达率之间无相关(r=-0.410,-0.143,P>0.05)。结论:TGF-β1介导的MMP-2和TIMP-2表达失衡在翼状胬肉的发生、发展及侵犯角膜过程中发挥着重要的作用。     

人TGF-β2特异性siRNA质粒的构建

目的:构建人TGF-β2特异性siRNA质粒。方法:从NCBI中查找人TGF-β2的mRNA序列,并利用生物信息学对序列进行分析;根据siRNA的设计原则,选择最佳的siRNA序列;利用限制性内切酶BamHⅠ和BbsⅠ将相应的双链DNA插入到GPU6/GFP/Neo载体中;重组质粒经限制性内切酶BamHⅠ和PstⅠ单酶切进行鉴定,鉴定正确的质粒进一步通过基因测序的方法进行验证。结果:根据生物信息学选择TGF-β2mRNA的1016～1034区域作为干扰位点;重组质粒酶切结果表明双链DNA序列成功插入到了GPU6/GFP/Neo载体中;测序分析结果进一步证明插入序列与设计完全一致。结论:成功构建了人TGF-β2特异性siRNA干扰质粒,为研究TGF-β2基因功能和利用基因治疗的方法提高青光眼滤过手术成功率奠定了实验基础。     

Inhibition of TGF-β2-induced migration and epithelial-mesenchymal transition in ARPE-19 by sulforaphane

AIM: To investigate the effects of sulforaphane (SFN) on transforming growth factor (TGF)-β2 stimulated migration and epithelial-mesenchymal transition (EMT) in ARPE-19 cells. METHODS: ARPE-19 cells were cultured in the presence or absence of SFN or TGF-β2. SFN toxicity was assessed by performing a lactate dehydrogenase assay (LDH) and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assays, and cell migration was evaluated by Transwell migration assay. Actin stress fiber formation in ARPE-19 cells was determined using immunofluorescence analysis. Immunoblotting analysis was used to determine fibronectin and α-smooth muscle actin expressions along with the degree of Smad and Akt phosphorylation. RESULTS: SFN inhibited ARPE-19 migration. Additionally, SFN attenuated TGF-β2-induced appearance of actin stress fibers as well as fibronectin and α-smooth muscle actin expressions in these cells. SFN also hindered the TGF-β2-stimulated phosphorylation of Smad2, Smad3, and Akt. SFN showed no cytotoxicity towards ARPE-19 cells. CONCLUSION: SFN inhibits TGF-β2-stimulated migration and EMT in ARPE-19 cells, probably by preventing the establishment of actin stress fibers and Akt and Smad2/3 signaling.     

Artesunate inhibits proliferation and migration of RPE cells and TGF-β2 mediated epithelial mesenchymal transition by suppressing PI3K/AKT pathway

AIM:To study the braking effectiveness of artesunate on transforming growth factor(TGF)-β2 mediated epithelial-mesenchymal transition(EMT)in retinal pigment epithelium(RPE)in vitro.METHODS:The fostered ARPE-19 cells were processed with artesunate alone or combined with the TGF-β2.The CCK-8 examination was utilized to test the cell propagation.Cell migration was detected by scratch as well as the Transwell examination.The EMT characters and activation of PI3K/Akt signal channel were estimated by Western blotting and immunofluorescence.The Western blotting was utilized in order to confirm the vitreous of controls as well as patients with proliferative vitreoretinopathy(PVR)were collected and the levels of PI3K,phospho-PI3K,Akt.RESULTS:Disposal of ARPE-19 cells with artesunate(50-150μmol/L)obviously suppressed their propagation and immigration,which dependent on the concentration and time.Artesunate suppressed the EMT which was induced by TGF-β2 in ARPE-19 cells through sustaining the expression of vimentin andα-SMA through the suppression of PI3K,phospho-PI3K,phospho-Akt and Akt.Levels of PI3K,phospho-PI3K,AKT and phospho-Akt was increased in the vitreous in PVR(P<0.05).CONCLUSION:Such findings indicate that PI3K/Akt signal channel is highly activated in vitreous of PVR.Artesuante is an operative depressor of the propagation,immigration and TGF-β2-mediated EMT of ARPE-19 cells by reduced the expression of PI3K/Akt channel.     

TGF-β2体外抑制牛角膜内皮细胞增殖的研究

目的:观察体外不同浓度的转化生长因子β2(transforming growth factor-β2,TGF-β2)对牛角膜内皮细胞(bovine corneal endothelial cells,bCECs)的抑制作用,探讨其可能的分子机制。方法:培养新生小牛的角膜内皮细胞,将第3代内皮细胞转移至96孔培养板,实验分6组,每组设定4个复孔。1,2组分别为只含有培养液不含细胞和TGF-β2的空白对照组,和只含细胞和培养液不含TGF-β2的阴性对照组,3～6组分别加入含0.1,1,10,100ng/LTGF-β2的培养液。作用24,48,72h后用MTT检测法测定各孔吸光度A值。作用48h后,用流式细胞检测仪对各组细胞周期进行检测。RT-PCR法测定不同浓度的TGF-β2组p27Kip1基因的表达情况。结果:TGF-β2浓度为0.1～1ng/L,作用24～72h,能明显的使bCECs的吸光度发生改变,作用48h抑制效果最明显。0.1和1ng/L浓度组吸光度与阴性对照组相比有显著差异性,作用48h可使bCECs的G0/G1周期细胞所占比例与阴性组对比有显著差异,其p27Kip1表达较其他组明显增强。结论:0.1～1ng/L TGF-β2作用48h对bCECs具有显著增殖抑制作用,其抑制作用可能是通过上调细胞周期蛋白p27来实现的。