甲氨蝶呤治疗类风湿性关节炎骨破坏的研究进展

目的:了解甲氨蝶呤(Methotrexate,MTX)在治疗类风湿性关节炎(Rheumatoid arthritis,RA)骨破坏方面的研究进展。方法:通过查阅文献,综述MTX抑制滑膜T细胞的增殖并且诱导其凋亡,抑制T细胞因子以及细胞黏附因子的表达,以及对RA滑膜血管生成的抑制作用。结果与结论:MTX通过抑制滑膜T细胞、T细胞因子及细胞黏附因子的表达,促进细胞凋亡,抑制滑膜血管异常新生,达到抑制RA骨破坏的作用。     

内皮抑素概论及抗肺癌作用

肿瘤的生长转移与肿瘤血管生成密切相关,抗血管生成是治疗肿瘤的新策略.内皮抑素是特异性的血管内皮细胞生长抑制因子,能显著抑制肿瘤血管增生并诱导肿瘤细胞凋亡.内皮抑素的抗血管生成治疗和血管靶向基因治疗,为治疗肺癌提供了广阔前景 .     

canstatin抗肿瘤作用研究现状

肿瘤的生长及转移均依赖于血管新生。抗血管生成治疗已成为肿瘤的治疗热点,目前以内源性血管生成抑制因子研究为主。血管能抑素（canstatin）是新近发现的一种来源于IV型胶原蛋白的内源性抗血管新生抑制因子,具有较强的抗血管新生作用而受到重视,现综述其抗肿瘤研究现状。     

重组腺病毒介导的内皮抑素抗关节炎大鼠滑膜血管新生机制的研究

目的研究重组腺病毒介导的人内皮抑素(rAD-GFP-ES)对大鼠Ⅱ型胶原诱导型关节炎(CIA)血管新生的影响。方法首先,扩增并纯化重组腺病毒。然后,建立CIA模型,自造模次日治疗组给予rAD-GFP-ES(1×1011pfu·kg-1·wk-1×4wk),在第4周取膝关节进行免疫组化检测其滑膜的微血管密度(MVD),取血清进行Western blot检测大鼠体内内皮抑素(ES)、血管内皮细胞生长因子(VEGF)、基质金属蛋白酶-3(MMP-3)的表达并计算相对含量。结果①感染rAD-GFP-ES的大鼠体内ES获得了稳定表达,是非模型组的2.4倍。②感染rAD-GFP-ES可使滑膜新生血管数目明显减少,同时能降低VEGF和MMP-3的表达。结论VEGF和MMP-3可能共同促进CIA大鼠滑膜血管新生的形成和发展,rAD-GFP-ES可能通过抑制VEGF和MMP-3的表达而发挥抗滑膜血管新生的作用。     

类风湿关节炎中血管生成的分子机制

类风湿关节炎患者滑膜中血管生成增加 ,血管增生是造成滑膜炎 ,血管翳生长 ,骨和软骨破坏及骨赘形成的原因。类风湿关节炎滑膜组织中有许多促血管生成因子被表达 ,它们在类风湿关节炎血管生成的过程中发挥了重要作用。因此抑制新生血管生成 ,尤其是抑制血管内皮生长因子 ,是治疗类风湿关节炎的新方法。     

肿瘤血管生成抑制剂

恶性肿瘤的发展与侵袭均依赖于新生血管生成。肿瘤新生血管的生成受多种调节因子作用,这些调控因子主要有：血管内皮生长因子、血管生成素,成纤维细胞生长因子,血管抑素,内皮抑素,thrombospondin(TSP-1)等。通过抑制肿瘤新生血管的生成可以阻止肿瘤的发展和转移,甚至使肿瘤消退。抗血管生成药物为肿瘤的治疗提供了一个全新的思路,本文简单讨论了肿瘤血管生成与调节机制并着重介绍肿瘤血管生成抑制剂的研究进展。     

血管抑素在肿瘤诊断与治疗中的作用

血管抑素是内源性血管生成抑制因子，能抑制血管内皮细胞的增殖和迁移，从而抑制肿瘤的生长。通过各种实验方法能够检测肿瘤中的血管抑素水平，并将其作为新生血管抑制药应用于肿瘤治疗中。血管抑素在未来的肿瘤诊断、预后判断和治疗中有着广阔的应用前景。     

抗肿瘤血管治疗的新进展

肿瘤的发生、生长和转移与肿瘤的新生血管的形成密切相关 ,调节肿瘤新生血管生成的因子主要有促进因子和抑制因子两大类。较为重要的促进因子有血管内皮细胞生长因子 (VEGF)、碱性成纤维细胞生长因子 (b FGF)、血管生成素 (angiopoi-etin)、整合素 (integrins)等 ,这些因子与受体结合发挥其促血管生成的作用 ,因此阻断这些因子和受体的结合以及受体后信号传导途径可以抑制血管的生成。较为重要的血管生成抑制因子有内皮抑素(endostatin)、血抑素 (angiostatin)、凝血酶敏感蛋白 (thrombospondin,TSP)、基质金属蛋白酶 (ma-trix metallop…     

内皮抑素与角膜新生血管

正常角膜组织透明,无血管,周围血管终止于角膜缘,形成血管网,营养成分由此扩散入角膜。无血管是角膜的主要特征,也是维持角膜透明的重要条件。在病理状态下,新生毛细血管由角膜缘处侵入角膜内,称为角膜新生血管（corneal neovascularization,CNV）。内皮抑素（endostatin）是一种新型内源性新生血管生成抑制因子,可特异性抑制新生血管内皮细胞的增殖,封闭新生血管的形成,在肿瘤实验过程中表现出极强的抑瘤活性。实验表明,内皮抑素对多种起源的新生血管内皮细胞增殖有抑制作用,而不影响静止的血管内皮细胞,内皮抑素具有明显抑制角膜新生血管生长的作用,而无毒副作用,为临床防治CNV提供了一种新的途径。     

重组人内抑素对佐剂性关节炎大鼠滑膜组织血管内皮细胞生长因子表达的影响

目的:观察重组人内抑素对佐剂性关节炎大鼠滑膜组织血管内皮细胞生长因子(vascular endo-thelial cell growth factor,VEGF)的表达及其作用。方法:采用弗氏完全佐剂(Freund’s complete adjuvant,CFA)诱导的佐剂性关节炎(adjuvant-induced arthri-tis,AA)大鼠模型,用足容积法测量关节肿胀度;免疫组织化学法检测血管内皮细胞生长因子的表达及其对微血管密度的影响。结果:弗氏完全佐剂致炎后d 10,佐剂性关节炎大鼠出现继发性炎症,皮下注射给予不同剂量的重组人内抑素(1.25、2.5、5 mg.kg-1),连续7 d,对照组于d 10给予氨甲喋呤(Methotrexate,MTX,1.0 mg.kg-1)。重组人内抑素各剂量组能明显抑制AA大鼠的继发性足肿胀,重组人内抑素各剂量组可不同程度减少AA大鼠关节滑膜组织高表达的VEGF,还可使滑膜组织的微血管密度减少。结论:重组人内抑素可显著抑制AA大鼠滑膜组织VEGF的表达及微血管密度,该作用可能与抑制血管翳的生成及减少滑膜细胞表面VEGF的表达有关。     

Anti-adjuvant arthritis of recombinant human endostatin in rats via inhibition of angiogenesis and proinflammatory factors

AIM: To investigate the profile of endostatin on adjuvant arthritis (AA) and angiogenesis blockade in synovitis. METHODS: The model of rat AA was induced by injection of intradermal complete Freund's adjuvant (CFA). Hind paw volume of rat was measured by volume meter and the activities of interleukin-1 (IL-1) and IL-2 were measured by the assay of thymocytes proliferation. IL-1beta and tumor necrosis factor-alpha (TNF-alpha) produced by synoviocytes was estimated with radioimmunoassay. The number of new blood vessels in knee joint synovium was counted under microscope by hematoxylin and eosin (HE) staining. RESULTS: The secondary inflammation of AA rats appeared on the 10th day after injection of CFA. The therapeutic administration of endostatin (0.1, 0.5, and 2.5 mg/kg/d, sc, plus 7 d) was given from that time (d 10). It was found that endostatin significantly inhibited the secondary paw swelling and the number of new blood vessels in the synovium of AA rats. Endostatin significantly decreased the production of IL-1 derived from both peritoneal macrophages and synoviocytes and IL-2 from splenocytes, especially at the dose of 2.5 mg/kg. This effect of endostatin also was seen on TNF-alpha produced by synoviocytes. CONCLUSION: The recombinant human endostatin had an inhibitory effect on rat AA, which was related to its anti-angiogenesis and inhibition of proinflammatory cytokines.     

New antiangiogenic strategies for the treatment of proliferative synovitis

Angiogenesis inhibition, which has been extensively studied for the treatment of various malignancies, is beginning to emerge as a new potential therapy for proliferative synovitis, particularly rheumatoid arthritis (RA). The rheumatoid pannus, the site of inflammation and joint destruction in the rheumatoid synovium, relies on the development of new vasculature to sustain its growth. A host of mediators have been shown to induce angiogenesis at the site of the inflamed synovium; these include vascular endothelial growth factor, fibroblast growth factor, integrin alpha(V)beta3, angiopoietin, prosta-glandin E1 and prostaglandin E2, and matrix metalloproteinases. In addition, hypoxia at the site of synovial inflammation contributes to angiogenesis stimulation. Several naturally-occurring inhibitors exist, such angiostatin and endostatin. There are a number of drugs undergoing study in the treatment of proliferative synovitis, which capitalise on the correlation between angiogenesis inhibition and the reduction of signs and symptoms of RA. Paclitaxel and an anti-integrin alpha(V)beta3 antibody, LM-609, are currently in clinical trials. Other drugs that may inhibit angiogenesis in RA include TNP-470 (formerly called AGM-1470), PPI-2458, PTK-787, bevacizumab and thalidomide. Many of these drugs have shown promise for the treatment of oncologic disorders, and are now being evaluated for the treatment of proliferative synovitis.     

血管内皮抑素与顺铂对Calu-6肺癌细胞联合作用的观察

目的:研究血管内皮抑素对人肺癌细胞的增殖抑制作用及机制,并观察其与顺铂(DDP)的联合作用。方法:经血管内皮抑素及DDP干预后,以MTT法检测其对肺癌细胞Calu-6的抑制、流式细胞术检测细胞凋亡、ELISA测定Bcl-2/Bax、sFas/sFasL的表达。结果:(1)血管内皮抑素具有抑制Calu-6细胞增殖的作用(P<0.05)并呈现时间依赖性、诱导Calu-6细胞凋亡,下调Bcl-2表达,但对Bax的表达无明显影响(P>0.05),未检测到sFas/sFasL的表达。(2)血管内皮抑素与DDP联合用药方案中,同时给药较单药DDP诱导凋亡及下调Bcl-2表达明显,序贯给药与DDP间无明显差异(P>0.05)。(3)2种血管内皮抑素(恩度、内皮抑素)的效应差异无统计学意义(P>0.05)。结论:(1)血管内皮抑素可抑制Calu-6细胞生长、诱导其凋亡,其机制与下调Bcl-2表达有关。(2)血管内皮抑素与DDP间有增效作用,联合用药方案中以同时给药效果最著。(3)各血管内皮抑素的作用机制相似。     

The SCID mouse--a novel experimental model for gene therapy in human rheumatoid arthritis

Rheumatoid arthritis (RA) is characterized by a progressive destruction of joints accompanied by synovial hyperplasia, inflammation and autoimmune phenomena. RA is a common disease, but current animal models resemble human RA only to a limited extent. As recent experimental approaches support the concept that T-cell-independent pathways may play a major role in RA, we developed a severe combined immunodeficient (SCID) mouse model to study the molecular and cellular interaction between RA synovium and cartilage. Both RA synovium as well as isolated RA synovial fibroblasts were able to invade and degrade normal human cartilage when coimplanted under the renal capsule of the SCID mice. Subsequently, we used this model to study the effects of retrovirus-based gene transfer of potentially inhibitory molecules into human RA synovial fibroblasts. Overexpression of interleukin (IL)-1 receptor antagonist reduced perichondrocytic cartilage degradation, but did not affect the invasiveness of RA synovial fibroblasts. Overexpression of tumor necrosis factor (TNF)-alpha receptor p55 revealed only a marginal effect. However, overexpression of IL-10 showed a most remarkable inhibition of cartilage destruction mediated by synovial fibroblasts. The SCID mouse model is a most useful tool not only to study the molecular basis of cartilage destruction, but also to evaluate novel approaches of gene therapy.     

重组人内抑素对佐剂性关节炎大鼠滑膜细胞增殖及产生细胞因子的影响

目的观察重组人内抑素对体外培养佐剂性关节炎大鼠滑膜细胞的增殖功能及产生细胞因子的影响,探讨其治疗佐剂性关节炎的作用机制。方法采用弗氏完全佐剂(CFA)诱导的佐剂性关节炎(AA)大鼠模型,用足容积法测量关节肿胀度;采用collagenasetypeⅡ消化法分离、培养滑膜细胞,MTT法检测重组人内抑素体内用药对滑膜细胞增殖的影响;放免法检测滑膜细胞上清液中IL-1β、TNF-α的含量。结果CFA致炎后d10,AA大鼠出现继发性炎症,此时皮下注射重组人内抑素(1·25,2·5,5mg·kg-1),连续7d,对照组于d10给予MTX(1·0mg·kg-1)。各剂量组能明显抑制AA大鼠的继发性足肿胀;重组人内抑素体内用药可明显减少AA大鼠滑膜细胞数量,抑制滑膜细胞的增殖,并呈剂量依赖关系;各剂量组均可明显抑制AA大鼠滑膜细胞产生过高的IL-1β和TNF-α。结论重组人内抑素抑制AA大鼠滑膜细胞过度的增殖及过高的细胞因子是其治疗AA的途径之一。     

Bee venom induces apoptosis through caspase-3 activation in synovial fibroblasts of patients with rheumatoid arthritis.

Bee venom (BV) has been used traditionally for the control of pain and inflammation in various chronic inflammatory diseases, including rheumatoid arthritis (RA) in Oriental medicine. However, it is still unclear how BV exerts its beneficial effects on the clinical course of RA patients. To investigate the effect of BV on the treatment of rheumatoid synovitis, we examined the inhibition of cell growth and induction of apoptosis in human rheumatoid synovial fibroblasts. Rheumatoid synovial fibroblasts were surgically obtained from patients with RA. Cell proliferation and viability were assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The apoptosis of synovial cells treated with 10 microg/ml BV for 24 h was identified by 4,6-diamidino-2-phenylindole (DAPI) staining, terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assay, DNA fragmentation assay, RT-PCR, and Western blot analysis. It was demonstrated that rheumatoid synovial cells treated with 10 microg/ml BV for 24 h exhibited apoptotic features and fragmentation of DNA. In addition, BV induces apoptosis in rheumatoid synovial cells through a decrease in BCL2 expression and an increase in BAX and caspase-3 (CASP3) expression. It is suggested that BV inhibits the proliferation of rheumatoid synovial cells through induction of apoptosis by CASP3 activation.     

Blockage of the formation of new blood vessels by recombinant human endostatin contributes to the regression of rat adjuvant arthritis

The formation of new blood vessels permits a supply of nutrients and oxygen to the proliferating synovial cells and augmented inflammatory cell mass in rheumatoid arthritis (RA). Angiogenesis inhibition is not dependent on a down-regulated immune system. Therefore, angiogenesis is an attractive target in treating rheumatoid arthritis. To confirm the effect of recombinant human endostatin, an angiogenesis inhibitor, on inflammatory angiogenesis and to elucidate the related mechanisms, rat adjuvant arthritis model induced by Freund's complete adjuvant was used. The secondary arthritis was evaluated by using clinical scores and determining the volume of hind paw swelling. The number of new blood vessels was counted under microscope based on HE (hematoxylin and eosin) staining and positive immunoreactivity of factor VIII related antigen. factor VIII related antigen and vascular endothelial growth factor (VEGF) expressions in synovial tissue were determined by using immunohistochemistry. It was found that endostatin attenuated rat secondary paw swelling induced by Freund's complete adjuvant in a dose-dependent manner. Meanwhile, the number of new blood vessels in synovial tissue stained with HE was reduced after treatment with endostatin, which was proved by the positive immunostaining of factor VIII related antigen. Further, endostatin decreased the expression of VEGF in both cartilage and synovial tissue. These suggest that endostatin inhibiting VEGF expression contributes to the regression of rat adjuvant arthritis.     

Fraxetin inhibits the induction of anti-Fas IgM, tumor necrosis factor-alpha and interleukin-1beta-mediated apoptosis by Fas pathway inhibition in human osteoblastic cell line MG-63

The survival of osteoblast cells is one of the determinants of the development of osteoporosis in patients with inflamed synovium, such as in rheumatoid arthritis (RA). By means of alkaline phosphatase (ALP) activity and osteocalcin ELISA assay, we have shown that fraxetin exhibits a significant induction of differentiation in the human osteoblast-like cell line MG-63. In addition, we also assessed whether fraxetin affects inflammatory cytokine-mediated apoptosis in osteoblast cells. TNF-alpha or IL-1beta enhance apoptotic DNA fragmentation in anti-Fas IgM-treated MG-63 cells by increasing Fas receptor expression. However, TNF-alpha or IL-1beta treatment alone does not induce apoptosis. Treatment of MG-63 cells with fraxetin not only inhibited anti-Fas IgM-induced apoptosis, but also blocked the synergetic effect of anti-Fas IgM with TNF-alpha or IL-1beta on cell death. The apoptotic inhibition of fraxetin is associated with inhibition of TNF-alpha and IL-1beta-mediated Fas expression and enhancement of FLIP expression, resulting in a decrease of caspase-8 and caspase-3 activation. These results indicate a potential use of fraxetin in preventing osteoporosis by inhibiting inflammatory cytokine-mediated apoptosis in osteoblast cells.     

Retinoic acid regulates cell cycle genes and accelerates normal mouse liver regeneration

All-trans retinoic acid (RA) is a potent inducer of regeneration. Because the liver is the principal site for storage and bioactivation of vitamin A, the current study examines the effect of RA in mouse hepatocyte proliferation and liver regeneration. Mice that received a single dose of RA (25 μg/g) by oral gavage developed hepatomegaly with increased number of Ki67-positive cells and induced expression of cell cycle genes in the liver. DNA binding data revealed that RA receptors retinoic acid receptor β (RARβ) and retinoid x receptor α (RXRα) bound to cell cycle genes Cdk1, Cdk2, Cyclin B, Cyclin E, and Cdc25a in mice with and without RA treatment. In addition, RA treatment induced novel binding of RARβ/RXRα to Cdk1, Cdk2, Cyclin D, and Cdk6 genes. All RARβ/RXRα binding sites contained AGGTCA-like motifs. RA treatment also promoted liver regeneration after partial hepatectomy (PH). RA signaling was implicated in normal liver regeneration as the mRNA levels of RARβ, Aldh1a2, Crabp1, and Crbp1 were all induced 1.5 days after PH during the active phase of hepatocyte proliferation. RA treatment prior to PH resulted in early up-regulation of RARβ, Aldh1a2, Crabp1, and Crbp1, which was accompanied by an early induction of cell cycle genes. Western blotting for RARβ, c-myc, Cyclin D, E, and A further supported the early induction of retinoid signal and cell proliferation by RA treatment. Taken together, our data suggest that RA may regulate cell cycle progression and accelerates liver regeneration. Such effect is associated with an early induction of RA signaling, which includes increased expression of the receptor, binding proteins, and processing enzyme for retinoids.