转化生长因子α和增殖细胞核抗原在原发性肝癌及癌旁肝组织中的表达

目的：探讨转化生长因子α（ＴＧＦα）和增殖细胞核抗原（ＰＣＮＡ） 在原发性肝癌（ＨＣＣ） 和癌旁肝组织的表达情况及其与肝组织增殖的关系。方法：用免疫组织化学方法检测ＴＧＦα和ＰＣＮＡ 在正常肝组织、原发性肝癌（ＨＣＣ）和癌旁肝组织的表达。结果：ＴＧＦα和ＰＣＮＡ在正常肝组织中均不表达;６１－０％ 的ＨＣＣ 癌组织和５７－５％ 的癌旁肝组织表达ＴＧＦα;１００ ％ 的癌组织和９７－５ ％ 的癌旁肝组织可观察到ＰＣＮＡ阳性反应。经统计学分析,癌组织的ＴＧＦα染色强度显著高于癌旁肝组织（Ｐ＜０－０１） ;ＴＧＦα阳性组癌组织和癌旁肝组织的ＰＣＮＡ 标记指数（ＬＩ） 均分别高于ＴＧＦα阴性组（ Ｐ均＜０－０１）。结论：ＨＣＣ癌组织和癌旁肝组织存在ＴＧＦα的异常表达;ＴＧＦα与ＨＣＣ癌组织和癌旁肝细胞的增殖密切相关。推测ＨＣＣ中可能存在正反馈的ＴＧＦα自分泌机制     

转化生长因子α和增殖细胞核抗原在原发性肝癌及癌旁肝组织 …

目的：探讨转化生长因子α（ＴＧＦα）和增殖细胞核原（ＰＣＮＡ）在原发性肝癌（ＨＣＣ）和癌旁组织的表达民政部及其与肝组织增殖的关系。方法：用免疫组织化学方法检测ＴＧＦα和ＰＣＮＡ在正常肝组织、原发性肝癌（ＨＣＣ）和癌旁肝组织的表达。结果：ＴＧＦα和ＰＣＮＡ在正常肝组织中均不表达;６１．０％的ＨＣＣ癌组织和５７．５％的癌旁肝组织表达ＴＧＦα;１００％的癌组织和９７．５％的癌旁肝组织可观察到ＰＣＮＡ阳性     

转化生长因子—β1及其受体的表达与大鼠肝纤维化关系 …

探讨肝纤维化形成过程转化生长因子－β及其受体的表达对胶原合成的影响。方法：采用免疫组化和斑点杂交技术,观察实验性大鼠肝纤维化过程肝内ＴＧＦ－β１,ＴＧＦ－βＲⅡ和Ⅰ、Ⅲ型前胶原ｍＲＮＡ及其蛋白表达的动态变化。结果随着肝纤维化程度的加重,肝内ＴＧＦ－β１、ＴＧＦ－βＲⅡ和Ⅰ、Ⅲ型前胶原ｍＲＮＡ及其蛋白表达均明显增加,组间比较均有显著差异（Ｐ〈０．０５或Ｐ〈０．０１）。ＴＧＦ－β１与Ⅰ、Ⅲ型前胶原ｍＲ     

低密度脂蛋白对肾小珠系膜细胞,TGF—β和FN mRNA表达的影响

目的：观察低密度脂蛋白（ＬＤＬ）对体外培养的大鼠肾小球系膜细胞（ＭＳＣ）生长及转化生长因子β（ＴＧＦ－β）和纤维连接蛋白（ＦＮ）基因表达的影响。方法：体外培养的ＭＳＣ培养液中加入ＬＤＬ共同孵育,采用＾３Ｈ－ＴｄＲ渗入法检测ＭＳＣ增殖情况,应用Ｎｏｒｔｈｅｒｎ ｂｌｏｔ检测ＴＧＦ－β ｍＲＮＡ、ＦＮ ｍＲＮＡ的表达,应用斑点杂交法检测抗ＴＧＦ－抗体对ＦＮ ｍＲＮＡ表达的影响。结果（１）ＬＤＬ刺激ＭＳ     

PDGF与肝素对动脉SMCI,Ⅲ型前胶原mRNA表达及胶原蛋白合成？…

目的 研究,探讨血小板源生长因子（ｐｌａｔｅｌｅｔｄｅｒｉｖｅｄｇｒｏｗｔｈｆａｃｔｏｒ,ＰＤＧＦ）和肝素对人主动脉平滑肌细胞（ｈｕｍａｎａｏｒｔａａｍｏｏｔｈｍｕｓｃｌｅ,ｃｅｌｌ,ｈＡＳＭＣ）增殖,胶原蛋白的合成,分泌以及Ｉ,Ⅲ型前胶原ｍＲＮＡ表达和转移生长因子β（ｔｒａｎｓｆｏｒｍｉｎｇｇｒｏｗｔｈｆａｃｔｏｒβ－ＴＧＦ－β）ｍＲＮＡ表达的调节作用,方法＾３Ｈ－ＴｄＲ,＾３Ｈ－脯氨酸掺入及Ｎ     

肝细胞生长因子对大鼠急性肝损伤保护作用的实验研究

目的：研究肝细胞生长因子（ＨＧＦ）对大鼠急性肝损伤的保护作用。方法：ＣＣｌ４所致大鼠肝损伤作为观察对象。ＨＧＦ治疗组分别与损伤组、正常对照组作比较。结果：治疗组血清ＡＬＴ、α－肿瘤坏死因子（ＴＮＦ－α）、肝组织匀浆ＭＤＡ、肝组织钙含量和血清及肝组织匀浆超氧化物歧化酶（ＳＯＤ）活力均有明显改善。结论：肝细胞生长因子对肝脏有保护作用。     

肝硬变患者TGF—β1基因在外周血中表达及与PⅢP血清水平？…

目的：为验证肝纤维化动物模型和慢性肝病患者肝组织中,转化生长因子（ＴＧＦ－β１）的ｍＲＮＡ表达增强。作者分析肝硬变患者外周血ＴＧＦ－β１ｍＲＮＡ和血清中有活性的ＴＧＦ－β１蛋白及其结果与Ⅲ型前胶原Ｎ端肽血清水平间的关系。方法：肝硬变患者５０自愿者８人,取其外周血,分离单核细胞（ＰＢＭＣ）,提取ＲＮＡ,用巢式ＲＴＰＣＲ扩增ＴＧＦ－β１ｍＲＮＡ并测定血清中ＴＧＦ－β１和ＰⅢＰ的水平。结果：分析表明患者     

大鼠浅Ⅱ度烫伤愈合渗出液细胞中生长因子mRNA的表达动态

为探讨生长因子在浅Ⅱ度烫伤愈合中的作用提供基础资料，从分子水平研究了烫伤愈合渗出液细胞中生长因子ｍＲＮＡ的表达动态。在ＳＤ大鼠背部埋入海绵，于热烫伤浅Ⅱ度后６ｈ和１、３、７、１０、１４ｄ分别取渗出液细胞，提取细胞总ＲＮＡ，用逆转录聚合酶链反应方法，以βａｃｔｉｎ为内对照，计算ＴＧＦα、ＴＧＦβ１、ＰＤＧＦ和ｂＦＧＦｍＲＮＡ表达量的变化。结果表明：伤后６ｈ，ＴＧＦα、ＴＧＦβ１、ｂＦＧＦ均有表达；伤后３ｄＴＧＦα、ＴＧＦβ１、ＰＤＧＦ均出现表达峰，表达水平明显高于对照组（Ｐ＜０．０５）；伤后１０ｄＴＧＦβ１、ｂＦＧＦ出现表达峰，表达水平也明显高于对照组（Ｐ＜０．０５）。这提示它们在烫伤愈合的不同时期协同发挥效应。     

转化生长因子αmRNA在大肠癌组织和转移中的表达及其临床意义

为探讨转化生长因子α（ＴＧＦ－α）ｍＲＮＡ在大肠癌发生、发展中的作用及其临床意义，应用核酸杂交方法对１４例大肠癌患者３组不同组织及直肠癌细胞系ＨＲ－８３４８进行ＴＧＦ－αｍＲＮＡ表达检测。结果显示：１４例大肠癌３组不同组织及细胞系中均有ＴＧＦ－αｍＲＮＡ不同程度的表达，以癌远侧组织中表达为最弱：ＴＧＦ－αｍＲＮＡ表达与Ｄｕｋｅｓ分期相关，可望作为判断大肠癌恶性程度的指标。另根据其在癌侧组织中表达减     

肝纤维化时Ⅳ型胶原酶与金属蛋白酶组织抑制因子-1的表达

目的 研究肝纤维化时Ⅳ型胶原酶（ＭＭＰ－２）和金属蛋白酶组织抑制因子－１（ＴＩＭＰ－１）在肝组织与贮脂细胞（ＦＳＣ）中的表达,探讨其对肝纤维化的作用。方法 分别从正常和ＣＣｌ４肝纤维化大鼠的肝组织及ＦＳＣ中提取ＲＮＡ,应用逆转录－聚合酶链反应（ＲＴ－ＰＣＲ）方法检测ＭＭＰ－２与ＴＩＭＰ－１ ｍＲＮＡ水平。结果 正常肝脏ＦＳＣ不表达ＭＭＰ－２,仅肝纤维化时才转录;肝组织中无论量中肝纤维化时均存在ＭＭ     

肝纤维化中丹参素对TGFβ1/Smads/ERK信号通路的影响及其相互关系

目的探讨肝纤维化过程中转化生长因子β1(transforming growth factorβ,TGFβ1)/Smads/细胞外信号调节激酶(extracellular signal-regulated kinase,ERK)通路的相互作用。方法体外分离培养大鼠肝星状细胞(hepatic stellatecell,HSC),MTT法筛选丹参素最适浓度;以最适浓度丹参素及ERK阻断剂(PD98059)作用于经TGFβ1处理的HSC,CCK-8法检测各组HSC增殖;免疫细胞化学染色法检测各组HSC内α-平滑肌肌动蛋白(α-smooth muscle sctin,α-SMA)及Ⅰ、Ⅲ型胶原表达;RT-PCR检测各组HSC的Smad2、Smad3、Smad7 mRNA表达量;Western blot法检测各组Smad2/3、ERK1/2蛋白磷酸化及Smad7蛋白水平。结果 0.062 5～0.25 mmol/L的丹参素可有效抑制HSC增殖,作为最适处理浓度;PD98059(100μmol/L)可抑制TGFβ1诱导的HSC增殖,丹参素剂量依赖性抑制TGFβ1诱导的HSC增殖(P<0.01);TGFβ1促进细胞内α-SMA及Ⅰ、Ⅲ型胶原表达,PD98059及丹参素降低TGFβ1诱导的α-SMA及Ⅰ、Ⅲ型胶原水平;PD98059可拮抗TGFβ1诱导的Smad2、Smad3、Smad7 mRNA的表达(P<0.05,P<0.01),丹参素下调Smad2、Smad3 mRNA水平,但上调Smad7 mRNA水平(P<0.01);PD98059及丹参素抑制TGFβ1诱导的Smad2/3、ERK1/2蛋白磷酸化(P<0.01),但对TGFβ1诱导的Smad7蛋白表达上调有不同效应(P<0.01)。结论 TGFβ1上调Smad2、Smad3 mRNA表达及蛋白磷酸化,进而诱导HSC增殖、活化及胶原合成,ERK通路可能参与HSC中TGFβ1诱导Smad基因表达及蛋白磷酸化过程。丹参素对TGFβ1/Smad/ERK信号通路具有抑制效应。     

Effects of ribozyme targeting platelet-derived growth factor receptor β subunit gene on the proliferation and apoptosis of hepatic stellate cells in vitro

Background Activation and proliferation of hepatic stellate cells (HSC) is essentially involved in the development and progression of hepatic fibrosis. The most potent growth factor for HSC is platelet-derived growth factor receptor (PDGF) and PDGF receptor β subunit (PDGFR-β) is the predominant signal transduction pathyway of PDGF which is overexpressed in activated HSC. This study investigated the cleavage activity of hammerhead ribozyme targeting PDGFR-β mRNA in HSC and the effect on biological characteristics of HSC.Methods Expression vector of anti-PDGFR-β ribozyme was constructed and transfected into rat activated HSC with lipofectamin. The positive cell clones were gained by G418 selection. The expression of PDGFR-β, α-smooth muscle actin, and typeⅠand type Ⅲ collagen were detected by using Northern blot, Western blot and immunocytochemical staining, respectively. The cell proliferation was determined with MTT colorimetric assay. The cell apoptosis was analyzed by using flow cytometry, acridine orange fluorescence vital staining and transmission electron microscopy.Results The expression of PDGFR-β at mRNA and protein level was markedly reduced in ribozyme-transfected HSC by 49%-57% (P&lt;0.05-0.01). The proliferation and α-smooth muscle actin expression of ribozyme-transfected HSC were significantly decreased (P&lt;0.05-0.01), and the type Ⅰ and type Ⅲ collagen synthesis were also reduced (P&lt;0.01). In addition, the proliferative response of ribozyme-transfected HSC to PDGF BB was significantly inhibited. Otherwise, the apoptotic cells were significantly increased in ribozyme-transfected HSC (P&lt;0.01), and typical apoptotic cells could be found under transmission electron microscopy.Conclusions The anti-PDGFR-β ribozyme effectively cleaved the target RNA and significantly inhibited its expression, which blocked the signal transduction of PDGF at receptor level, inhibited HSC proliferation and collagen synthesis, and induced HSC apoptosis. These results suggest that inhibiting PDGFR-β expression of HSC may be a new target for the therapy of liver fibrogenesis, and ribozyme may be a useful tool for inhibiting PDGFR-β expression.     

一种胶质瘤相关的酸性成纤维细胞生长因子mRNA同源物

目的 克隆胶质瘤相关基因。方法 取胶质瘤患者新鲜肿瘤标本及肿瘤附近正常脑组织提取mRNA,反转录成cDNA,采用基于PCR的消减杂交法获得差异的cDNA,并经多聚酶链反应扩增后克隆人T载体测序,用打点杂交法验证差异克隆。结果 获得一胶质瘤相关的酸性成纤维生长因子基因同源物。结论 该酸性成纤维生长因子基因同源物是酸性成纤维生长因子mRNA的不同拼接产物,可能参与胶质瘤的形成。     

过氧化物酶增殖物激活受体γ抑制大鼠肝星状细胞中转化生长因子-β1诱导的结缔组织生长因子表达

目的 探讨过氧化物酶增殖物激活受体γ(PPARγ)对大鼠肝星状细胞(HSC)中转化生长因子-β1(TGF-β1)诱导的结缔组织生长因子(CTGF)表达的抑制作用.方法 常规培养大鼠HSC,预先给予或不给予PPARγ特异性拮抗剂GW9662处理,再经系列浓度的PPARγ天然配体(15-d-PGJ2)或合成配体(GW7845)及TGF-β1序贯作用后,通过半定量RT-PCR及Western-blotting分析CTGF表达状况,透射电镜观察HSC形态学变化.结果 15-d-PGJ2和GW7845可显著抑制HSC中TGF-β1诱导的CTGF表达(同时在转录和转录后水平),且PPARγ配体对CTGF表达的抑制作用可被GW9662部分或完全逆转,说明此种抑制效应确由PPARγ所介导;电镜观察亦显示HSC由活化态向静息态的形态学转变.结论 PPARγ活化可显著抑制HSC中TGF-β1诱导的CTGF表达,提示PPARγ可作为逆转肝纤维化治疗的新的有效靶点.

Abstract：

Objective To investigate the effect of peroxisome proliferator-activated receptor gamma(PPARγ)activation on transforming growth factor betal(TGF-β1)-induced connective tissue growth factor(CTGF)expression in rat hepatic stellate cells(HSCs).Methods Cultured HSCs with or without PPARγ-specific antagonist GW9662 treatment prior to the addition of an increasing amount of PPARγ natural ligand(15-d-PGJ2)or synthetic ligand(GW7845)were stimulated with TGF-β1.The mRNA and protein levels of CTGF expression were detected by semi-quantitative RT-PCR and Western blotting,respectively.The morphological changes of the HSC were obgerved by electron microscope.Results 15-d-PGJ2 and GW7845 significantly inhibited TGF-β1-induced CTGF expression at both mRNA and protein levels in HSCs, and the inhibitory effect was dramatically,if not completely,abolished by pretreatment with GW9662,suggesting that the inhibition was mediated by PPARγ. Morphological observation revealed that PPARγactivation caused obvious changes of HSCs from activated to quiescent phenotypes.Conclusion PPARγ ligand shows potent inhibitory effect on TGF-β1-induced CTGF expression in rat HSCs,suggesting its potential as a candidate agent for treatment and prevention of hepatic fibrosis.     

转化生长因子β1通过Smad2途径调节足细胞结缔组织生长因子表达

目的　研究肾脏足细胞是否表达结缔组织生长因子 (CTGF)以及TGFβ1对CTGF表达调控的信号途径。方法　以肾小球足细胞为对象 ,应用Western印迹分析技术 ,观察了 3种促进肾脏纤维化的蛋白因子 ,转化生长因子 β1(TGFβ1)、血小板源生长因子 (PDGF)和血管紧张素Ⅱ (AngⅡ )对体外培养的足细胞CTGF蛋白表达的影响 ,以及ERK、Smad两条信号途径在TGFβ1调节CTGF蛋白表达中的影响 ;逆转录 聚合酶链反应 (RT PCR)检测CTGFmRNA的变化。结果　体外培养的足细胞表达基础水平CTGF蛋白 ,2 0ng/mlPDGF和 10 -6mol/LAngII刺激 2 4小时后细胞内CTGF蛋白水平与对照相比差异无显著意义 (P >0 0 5 ) ,而 1ng/mlTGFβ1刺激 2 4h足细胞CTGF蛋白水平显著高于对照 (P <0 0 5 ) ,且增加呈TGFβ1剂量依赖趋势 ;1ng/mlTGFβ1刺激 12h可以使细胞CTGFmRNA表达增加。1ng/mlTGFβ1使足细胞Smad2 和细胞外信号调节激酶 (ERK1/ 2 )磷酸化 ,在刺激 30min达高峰 ;应用丝 /苏氨酸激酶抑制剂Staurosporine抑制Smad2 磷酸化可以消减TGFβ1刺激的CTGF蛋白增加 ,但ERK1/ 2 活化抑制剂PD 980 5 9阻断ERK1/ 2 磷酸化不能减弱TGFβ1刺激CTGF蛋白表达的效应。结论　在足细胞上 ,TGFβ1刺激CTGF表达依赖于Smad2 信号通路的活化 ,而不依赖于ERK1/     

Inhibition of the activation and collagen production of cultured rat hepatic stellate cells by antisense oligonucleotides against transforming growth factor-beta 1 is enhanced by cationic liposome delivery]

In order to investigate the inhibition of the activation and collagen production of cultured rat hepatic stellate cells (HSC) by antisense oligonucleotides (ASON) against TGF beta 1 after cationic liposome (lipofectin) delivery, the authors synthesized a 20-mer phosphorothioate antisense oligonucleotide of TGF beta 1 mRNA, and its sense of missense oligonucleotides, and then treated the HSC with cationic liposome/oligonucleotide complexes respectively. The cellular uptake of 32P-labelled oligonucleotides was determined by liquid scintillation counting, and HSC activation was assessed by the expression of alpha-smooth muscle actin(alpha-SMA). The cellular uptake of lipofectin/32P-ASON complex was approximately five-fold higher than that of 32P-ASON alone. Cationic liposome (lipofectin) delivery significantly increased the inhibition of HSCs activation by ASON, while compared with the use of naked TGF beta 1 ASON at the same concentration (at a final concentration of 1 mumol/L, P < 0.05). However, these effects were not observed in lipofectin alone, liposome/sense or missense oligos complexes at the same concentration. The oligonucleotide or lipofectin/oligos complexes had no cytotoxicity to rat HSCs in culture. These findings suggest that cationic liposome is an effective vehicle to improve the delivery of ASON to rat HSCs in culture, and the cationic liposome/TGF beta 1 ASON complex may be useful for the treatment of hepatic fibrosis.     

干扰素α对实验大鼠肝纤维化的治疗作用

目的探讨干扰素α(IFNα)对肝纤维化的治疗作用.方法雄性SD大鼠40只,采用复合因素造成肝硬化模型,随机取12只作为A组(模型组),余大鼠分为B组(IFNα治疗组)12只,每日肌注IFNα 1×105U共6W和C组(对照组)12只,每日肌注生理盐水共6W.取肝脏组织行光镜和电镜下观察病理变化.结果 IFNα治疗组肝纤维化程度明显减轻,激活状态的星状细胞(HSC)数量明显减少,并出现凋亡现象.结论 IFNα对肝纤维化有治疗作用.     

PDGFR-α在继发性骨髓纤维化骨髓组织中表达的研究

目的:探讨血小板衍化生长因子受体-ɑ(PDGFR-ɑ)在继发性骨髓纤维化骨髓组织中的表达及临床意义。方法:应用免疫组化染色法,检测继发性骨髓纤维化组、对照组骨髓组织中PDGFR-α的表达情况。结果:继发性骨髓纤维化组骨髓组织中PDGFR-α的表达明显高于对照组,差异有统计学意义(P<0.05)。结论:PDGFR-α可能与继发性骨髓纤维化的发生、发展存在一定关联。     

人酸性成纤维细胞生长因子在大肠杆菌的高效表达

目的：探讨人酸性成纤维细胞生长因子（acidic fibroblast growth factor, aFGF）在大肠杆菌中高效表达的方法及重组人aFGF的生物学活性。方法：①通过PCR法扩增人aFGF引入点突变,对其密码子进行改造;②构建aFGF-pET-28a重组质粒并在大肠杆菌BL21(DE3)中表达;③重组蛋白的纯化及生物学活性研究。结果：通过PCR扩增,将设计的核酸水平突变引入到aFGF DNA中,使其自起始密码ATG后的前50个碱基均为原核细胞偏爱密码子、并保持原氨基酸序列不变,成功地实现了重组人aFGF在大肠杆菌中的高效表达,并通过降低培养温度使大肠杆菌中可溶性目的蛋白的含量大幅增加,原核表达的aFGF亦具有天然生物学活性。结论：通过对aFGF DNA碱基进行改造,可大幅度提高aFGF在大肠杆菌中的表达量,且目的蛋白具有良好的生物学活性。