CTGF与FGF在促成纤维细胞增殖过程中的基因反应差异

结缔组织生长因子(CTGF)是某些内皮细胞即刻早期基因反应产物,其与FGF具有类似的促进成纤维细胞(KMB-17)增殖的功能;在此促增殖过程中CTGF和FGF所诱导的基因反应有所差异,CTGF诱导细胞表达c-myc,而FGF促进c-fos表达增加;此外两种因子均诱导与酪氨酸磷酸化过程密切相关的src基因表达,免疫沉淀证实CTGF结合细胞表面受体后可诱导细胞内相应蛋白的酪氨酸磷酸化.     

结缔组织生长因子对血管生成和细胞功能的调节

结缔组织生长因子(CTGF)是CCN家族的主要成员,是研究较多的CCN蛋白之一。CTGF能促进细胞的增殖、存活、迁移和黏附,调节血管生成分子(bFGF,VEGF)以及一些影响胞外基质完整性和稳定性的分子(胶原、MMPs和TIMPs)的活性。CTGF可以在多个控制位点采取直接或间接的机制调控细胞功能和血管生成。结合新的发现和新的视点,阐述CTGF对细胞功能和血管生成的调控作用以及与肿瘤生长的关系。     

转化生长因子-β1通过促进结缔组织生长因子表达抑制胰腺癌细胞PANC-1增殖

目的:研究转化生长因子-β1(Transforming growth factor-β1,TGF-β1)和结缔组织生长因子(connective tissue growth factor,CTGF)对人胰腺癌细胞系PANC-1增殖的影响。方法:首先利用MTT检测重组TGF-β1、CTGF及anti-CTGF抗体处理PANC-1细胞后,细胞的增殖状态,用流式细胞仪(Flow cytometer,FCM)检测转染pcDNA3.1(-)-CTGF后PANC-1细胞的凋亡情况,最后利用RT-PCR检测TGF-β1作用PANC-1细胞后CTGF mRNA的表达。结果:TGF-β1和CTGF均呈浓度依赖性抑制PANC-1细胞增殖,加入anti-CTGF抗体可以取消TGF-β1对PANC-1细胞增殖的抑制作用;FCM结果显示pcDNA3.1(-)-CTGF质粒转染促进PANC-1细胞凋亡;RT-PCR则证明TGF-β1促进PANC-1细胞中CTGF mRNA表达。结论:TGF-β1可能通过促进CTGF表达来发挥抑制胰腺癌细胞PANC-1增殖的作用。     

氯沙坦对糖尿病肾病UALB、TGF-β1和CTGF影响

目的:探讨氯沙坦对早期糖尿病肾病(DN)患者转化生长因子TGF-β1、结缔组织生长因子(CTGF)水平的影响。方法:70例早期DN患者随机分组,对照组(35例)采用糖尿病(DM)常规治疗,治疗组(35例)在常规治疗基础上加用氯沙坦,疗程2个月。采用酶联免疫吸附法(ELISA)测定两组患者治疗前后尿TGF-β1、CTGF水平,同时观察血压、胆固醇(TC)、甘油三酯(TG)、高密度脂蛋白(HDL)、低密度脂蛋白(LDL)、空腹血糖、及餐后2h血糖、BUN、血肌酐(Scr)、24h尿白蛋白(UALB)等指标的变化。结果:与治疗前比较,患者治疗后尿TGF-β1和CTGF水平下降(P<0.05),UALB降低,血压、空腹血糖、餐后2h血糖、Scr、BUN指标改善(P<0.05),对照组治疗后尿TGF-β1升高(P<0.05),CTGF变化不大;后上述指标改善不如观察组。结论:氯沙坦可通过降低DN患者TGF-β1、CTGF水平而延缓DN的发展。     

结缔组织生长因子在胰腺组织纤维化中的作用及机制探讨

目的 观察胰腺纤维化后结缔组织生长因子(Connective tissue growth factor,CTGF)在胰腺组织内的表达;进一步研究参与CTGF作用于胰腺星状细胞(pancreatic stellate cells,PSCs)的分子信号调控通路.方法 建立大鼠胰腺纤维化动物模型,HE染色、天狼猩红染色和免疫组织化学染色等方法观察胰腺纤维化后PSCs的活化情况及CTGF在胰腺组织的表达.Real-time RT PCR检测CTGF的基因表达.Western Blot检测PSCa内α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)及胶原蛋白Ⅰ(CollagenⅠ)水平.结果 胰腺组织纤维化后,PSCs大量活化,并显著表达CTGF.CTGF作用后,PSCs内CTGF mRNA、α-SMA和Collagen Ⅰ的合成均有显著增加,在给予不同的细胞信号通路阻断剂后,PSCs内α-SMA的合成有显著下降,而Collagen Ⅰ的降低没有表现出统计学差异.结论 CTGF参与了胰腺纤维化的调控,MAPK和PI3-K信号通路均参与了CTGF的调控作用.     

pcDNA3.1(+)-CTGF真核表达质粒构建及其在人成骨样细胞SaOS-2细胞中的表达*

目的:构建结缔组织生长因子(CTGF)的pcDNA3.1(+)真核表达质粒(pcDNA3.1(+)-CTGF),并检测其在人成骨样细胞SaOS-2中的表达,为进一步研究CTGF基因在骨发育和骨修复中的机制提供技术支撑。方法:采用PCR方法体外克隆CTGF基因全序列,将其用同源重组技术连接到线性pcDNA3.1(+)载体上,构建pcDNA3.1(+)-CTGF真核表达质粒,并对该质粒进行测序鉴定;鉴定无误后转染至SaOS-2细胞中,观察其48 h的表达情况。结果:基因测序证实pcDNA3.1(+)-CTGF真核表达重组质粒构建成功,与对照组相比,转染SaOS-2细胞48 h后的CTGF表达水平显著上调,达到对照组的4.8×10⁵倍(P＜0.01)。结论:成功构建了pcDNA3.1(+)-CTGF真核表达质粒,并能在人成骨样细胞SaOS-2中稳定表达,为深入研究CTGF基因对骨生成的调控机制奠定了基础。     

川芎嗪对转化生长因子β1刺激肝星状细胞CTGF及Ⅰ型胶原表达的影响

研究川芎嗪对大鼠肝星状细胞株(hepatic stellate cell-T6,HSC-T6)结缔组织生长因子CTGF(connective tissue growth factor)及Ⅰ型胶原表达的影响.培养HSC-T6细胞,不同浓度川芎嗪与转化生长因子-β1(transforming growth factor-β1,TGF-β1)刺激的HSC-T6共同孵育.用MTT(3-(4,5-Dimethyhhiazol-2-y1)-2,5-diphenyhetrazolium bromide)法检测HSC-T6增殖:免疫细胞化学法观察川芎嗪对CTGF表达的影响;Western印迹法检测CTGF蛋白的表达.EUSA(enzyme linked immunosorbent assay)法测定Ⅰ型胶原表达.结果表明,一定浓度(100、200、400、600 mg/L)川芎嗪能抑制HSC-T6增殖,且呈剂量依赖性.免疫细胞化学结果显示随着川芎嗪浓度的升高,CTGF表达依次递减,与正常对照组比较,具有显著性差异(t=4.216,P<0.01).川芎嗪还可以明显抑制CTGF蛋白的表达,并可抑制Ⅰ型胶原合成,二者抑制程度呈正相关关系(r=0.861,P<0.01).川芎嗪可能通过抑制HSC-T6细胞增殖,下调CTGF的表达,阻断Ⅰ型胶原合成,从而发挥其抗肝纤维化的作用.     

抗结缔组织生长因子特异性小干扰RNA的设计、合成及筛选

目的 设计和合成针对抗肝纤维化关键基因抗结缔组织生长因子（connective tissue growth factor,CTGF）的特异性小干扰RNA（siRNA）,并筛选高效的CTGF siRNA抗肝纤维化序列.方法 参照siRNA设计原则,应用RNA在线设计软件,设计3段RNA干扰候选序列,分别转染正常干细胞株（L-02）,以转染非特异性siRNA（与CTGF mRNA无同源性）作为对照,应用Western 印迹检测L-02细胞CTGF蛋白质表达.结果 与对照相比,转染siRNA的L-02细胞CTGF蛋白表达明显下调,且siCTGF-1、siCTGF-2、siCTGF-3各片段干扰率分别为44.91 %、93.99 %、81.34%,其中以siCTGF-2干扰率最高,效果最明显.转染非特异性siRNA的L-02细胞CTGF蛋白表达无明显变化（P＜0.05）.结论 不同的CTGF siRNA对L-02细胞CTGF表达水平具有不同的干扰效能,成功合成能高效阻抑CTGF表达的CTGF siRNA,为进一步探索更特异、更有效的抗肝纤维化基因治疗这一新途径打下坚实基础.     

高糖刺激下大鼠肾小球系膜细胞CTGF和MT1-MMP的表达

目的观察高糖刺激的大鼠肾小球系膜细胞结缔组织生长因子(CTGF)和膜型基质金属蛋白酶-1(MT1-MMP)的动态变化,探讨糖尿病肾病(DN)的发病机制。方法体外培养的大鼠HBZY-1肾小球系膜细胞分为正常糖对照组,高糖组和甘露醇高渗对照组,采用RT-PCR及Western印迹法分别检测CTGF和MT1-MMP的mRNA及蛋白的表达,用酶联免疫吸附法(ELISA)检测培养上清中IV型胶原的含量。结果与对照组相比,高糖组各时间点系膜细胞CT-GF表达明显上调,IV型胶原的分泌增加,且二者随时间持续增高;而MT1-MMP的表达则随时间呈明显下降趋势。结论高糖可诱导肾小球系膜细胞CTGF表达增加,同时抑制MT1-MMP的表达,二者可能参与DN中细胞外基质(ECM)代谢失衡过程。     

枸杞多糖对肝癌患者血清AFP、PHCA、VEGF及CTGF水平的影响

目的:探讨枸杞多糖对原发性肝癌患者血清中甲胎蛋白(AFP)、人肝癌抗原(PHCA)、血管内皮生长因子(VEGF)及结缔组织生长因子(CTGF)水平的影响。方法:选取我院诊治的原发性肝癌患者84例为研究对象,根据治疗方案分为两组,其中对照组41例给予肝动脉化疔栓塞术(TACE)治疗,实验组43例在对照组基础上给予枸杞多糖治疗。用酶联免疫吸附法对两组患者治疗前后血清中VEGF、CTGF及PHCA水平进行检测,应用电化学发光法对AFP进行检测,并比较两组患者的临床疗效。结果:干预前,两组的AFP以及PHCA水平无显著差异(P0.05);干预后,实验组AFP及PHCA水平显著低于对照组,差异具有统计学意义(P0.05);干预后,实验组患者VEGF和CTGF下降幅度显著明显高于对照组,差异具有统计学意义(P0.05);干预后,实验组治疗有效率(76.74%)显著高于对照组(60.98%),差异具有统计学意义(P0.05)。与同期对照组比较,实验组生存率较高,不良反应发生率较低,差异具有统计学意义,P0.05。结论:枸杞多糖能够降低原发性肝癌患者的血清AFP、PHCA、VEG以及CTGF水平,提高原发性肝癌治疗效果。     

The role of connective tissue growth factor, a multifunctional matricellular protein, in fibroblast biology.

Connective tissue growth factor (CTGF, CCN2), a member of the CCN family of proteins, is a cysteine-rich proadhesive matricellular protein that plays an essential role in the formation of blood vessels, bone, and connective tissue. As expression of this protein is potently induced by transforming growth factor-beta (TGFbeta), it has been hypothesized that CTGF mediates several of the downstream actions of TGFbeta. In particular, CTGF is profibrotic, as CTGF is overexpressed in fibrotic disease and synergizes with TGFbeta to promote sustained fibrosis in vivo. Over the last several years, key data regarding the developmental role and structure and function relationship of CTGF have emerged. In addition, increased information concerning the mechanisms underlying the control of CTGF expression in normal and fibrotic cells and the signal transduction pathways through which CTGF acts on cells has been uncovered. This review summarizes the current state of knowledge regarding CTGF biology.     

Genetic Analysis of Connective Tissue Growth Factor as an Effector of Transforming Growth Factor β Signaling and Cardiac Remodeling

The matricellular secreted protein connective tissue growth factor (CTGF) is upregulated in response to cardiac injury or with transforming growth factor β (TGF-β) stimulation, where it has been suggested to function as a fibrotic effector. Here we generated transgenic mice with inducible heart-specific CTGF overexpression, mice with heart-specific expression of an activated TGF-β mutant protein, mice with heart-specific deletion of Ctgf, and mice in which Ctgf was also deleted from fibroblasts in the heart. Remarkably, neither gain nor loss of CTGF in the heart affected cardiac pathology and propensity toward early lethality due to TGF-β overactivation in the heart. Also, neither heart-specific Ctgf deletion nor CTGF overexpression altered cardiac remodeling and function with aging or after multiple acute stress stimuli. Cardiac fibrosis was also unchanged by modulation of CTGF levels in the heart with aging, pressure overload, agonist infusion, or TGF-β overexpression. However, CTGF mildly altered the overall cardiac response to TGF-β when pressure overload stimulation was applied. CTGF has been proposed to function as a critical TGF-β effector in underlying tissue remodeling and fibrosis throughout the body, although our results suggest that CTGF is of minimal importance and is an unlikely therapeutic vantage point for the heart.     

Angiogenesis is not impaired in connective tissue growth factor (CTGF) knock-out mice.

Connective tissue growth factor (CTGF) is a member of the CCN family of growth factors. CTGF is important in scarring, wound healing, and fibrosis. It has also been implicated to play a role in angiogenesis, in addition to vascular endothelial growth factor (VEGF). In the eye, angiogenesis and subsequent fibrosis are the main causes of blindness in conditions such as diabetic retinopathy. We have applied three different models of angiogenesis to homozygous CTGF(-/-) and heterozygous CTGF(+/-) mice to establish involvement of CTGF in neovascularization. CTGF(-/-) mice die around birth. Therefore, embryonic CTGF(-/-), CTGF(+/-), and CTGF(+/+) bone explants were used to study in vitro angiogenesis, and neonatal and mature CTGF(+/-) and CTGF(+/+) mice were used in models of oxygen-induced retinopathy and laser-induced choroidal neovascularization. Angiogenesis in vitro was independent of the CTGF genotype in both the presence and the absence of VEGF. Oxygen-induced vascular pathology in the retina, as determined semi-quantitatively, and laser-induced choroidal neovascularization, as determined quantitatively, were also not affected by the CTGF genotype. Our data show that downregulation of CTGF levels does not affect neovascularization, indicating distinct roles of VEGF and CTGF in angiogenesis and fibrosis in eye conditions.     

Hydrogen peroxide is a novel inducer of connective tissue growth factor.

Connective tissue growth factor (CTGF) has recently been described as a fibrogenic factor and is greatly induced by various extracellular stimuli, such as transforming growth factor-beta (TGF-beta), dexamethasone, and serotonin. CTGF induces collagen type I and fibronectin, and the deposition of such molecules leads to fibrotic disease in many tissues. Intracellular reactive oxygen species (ROS) are generated by extracellular stress conditions and are produced as by-products of cellular metabolism. Imbalanced cellular redox status is a potent pathogenic factor that leads to various degenerative diseases, including tissue fibrosis. Since CTGF is believed to play a crucial role in fibrotic disease formation in many tissues, we examined the role of ROS in CTGF gene expression in human lens epithelial cell line B3. The results showed that CTGF was induced by reactive oxygen species such as hydrogen peroxide and hydroxyl radicals. Next, we examined whether CTGF induction by ROS is via newly synthesized TGF-beta. The results showed that ROS directly induced CTGF mRNA not via the increased TGF-beta synthesis or activation. Next, we treated AG490, which is the well-known inhibitor of Janus kinase (JAK), with hydrogen peroxide. AG490 abrogated the CTGF induction by ROS in a dose-dependent manner. The results suggest that JAK-2/-3 seems to be involved in the enhanced CTGF mRNA expression by hydrogen peroxide. In this report, we present that hydrogen peroxide is a novel inducer of CTGF gene expression and that JAK-2/-3 activation seems to play a role in CTGF induction.     

Connective tissue growth factor is secreted through the Golgi and is degraded in the endosome.

Connective tissue growth factor (CTGF) is a cysteine-rich heparin-binding polypeptide that promotes proliferation, collagen synthesis, and chemotaxis in mesanchymal cells. When coinjected subcutaneously with transforming growth factor beta (TGFbeta), CTGF promotes sustained fibrosis in rats. However, little is known about the cell biology and structure/functional relationship of CTGF. In particular, no detailed characterization of the subcellular localization of CTGF has occurred, nor have sequences been identified within this protein required for this localization. In this report, using immunofluorescence and Western blot analysis, we show that CTGF is localized to the Golgi apparatus both in dermal fibroblasts and activated hepatic stellate cells. Using these methods, no CTGF was detected in endosomal, plasma membrane, cytosolic or nuclear fractions. Addition of brefeldin A, a drug that disrupts the Golgi, blocks the secretion of CTGF. We further show that the amino-terminal 37 amino acids of CTGF are sufficient to localize a heterologous protein (red fluorescent protein, RFP) to the Golgi. Although within this region of human CTGF is a N-glycosylation site, tunicamycin, which blocks N-linked glycosylation, has no significant effect on CTGF secretion. Surprisingly, mutation of a single amino acid residue, CYS-34, to alanine prevents localization of a CTGF-RFP fusion protein to the Golgi. These results are the first proof that endogenous CTGF is localized to the Golgi apparatus. Furthermore, using exogenously added (125)I-labeled CTGF, we show that CTGF is internalized and rapidly degraded in the endosome. That is, CTGF is quantitatively secreted through the golgi and is degraded in the endosome.     

Connective tissue growth factor expression in human intervertebral disc: implications for angiogenesis in intervertebral disc degeneration

Intervertebral disc (IVD) degeneration is strongly associated with chronic low back pain, one of the most common causes of morbidity in the West. While normal healthy IVD is avascular, angiogenesis is a constant feature of IVD degeneration and has been shown to be associated with in-growth of nerves. Connective tissue growth factor (CTGF) plays a pivotal role in angiogenesis. To investigate the expression of CTGF in both normal and degenerated IVD, 21 IVDs were obtained from patients at surgery or postmortem examination and grouped according to the severity of histological degeneration. The immunohistochemical expression of CTGF was correlated with the degree of degeneration. CD31 immunohistochemistry was used to correlate IVD degeneration with vasculature. Our results showed that CTGF is expressed in non-degenerated and degenerated human IVDs and increased expression of CTGF is associated with degenerated discs, particularly within areas of neovascularization. We suggest that CTGF may play a role in angiogenesis in the human degenerated IVD.     

Transforming growth factor-beta/connective tissue growth factor axis in the kidney

Transforming growth factor-beta(1) (TGFbeta(1)) is recognized as both a fibrogenic and inflammatory cytokine and plays a critical role in the kidney pathophysiology. The dysregulation of TGFbeta(1) has been linked with the development of diabetic nephropathy. Connective tissue growth factor (CTGF) is a fibrogenic cytokine and is recognized as a downstream mediator of TGFbeta(1) in kidney fibrosis. TGFbeta(1) is involved in immunomodulation and fibrosis in the kidney. However, CTGF plays a more specific role in the fibrogenic pathways in the kidney proximal tubule cells. Moreover, CTGF facilitates TGFbeta(1) signaling and promotes renal fibrosis. This suggests CTGF could be a potential target for kidney fibrosis. Long-term inhibition and targeting TGFbeta(1) directly is problematic, therefore, a more fruitful direction targeting diabetic nephropathy may involve the development of therapeutic strategies specifically targeting CTGF.