肝素化胶原/壳聚糖多孔支架的制备及其血管化的研究

本研究旨在构建一种能快速血管化的人工真皮替代物。用冻干法制备了胶原／壳聚糖多孔支架，并对其进行肝素化，观察此支架的结构特征、亲水性、体外降解性和组织相容性，同时将血管生成素引入到此支架，对复合有血管生成素的肝素化支架的体内血管化进行了初步研究。结果表明，肝素化胶原／壳聚糖多孔支架具有合适的三维多孔结构、良好的吸水性和较理想的酶解稳定性，体内实验表明，此支架具有良好的组织相容性，血管生成素可加快支架的血管化。     

Gene therapy of amyotrophic lateral sclerosis

Two-year experiments were performed to evaluate the neurotrophic effect of hypoxia-inducible factors (vascular endothelial growth factor and angiogenin) expressed in recombinant human adenoviruses in amyotrophic lateral sclerosis. Randomized placebo-controlled trial demonstrated safety and good tolerability of the recombinant antiviral drugs. The life span of patients under conditions of hypoxia increased after treatment with the test drug, which was probably related to improved resistance of motoneurons. The presence of virus-neutralizing antibodies decreases the effectiveness of adenoviral vectors, which necessitates differential approach to the selection of patients and continuous monitoring of gene therapy. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 145, No. 4, pp. 467–470, April, 2008     

血管形成素在宫颈鳞癌组织中的表达

目的：观察血管形成素（angiogenin,ANG）在宫颈鳞癌组织中的表达情况并探讨其临床病理意义。方法：应用免疫组化方法对48例石蜡包埋宫颈鳞癌组织切片进行ANG蛋白表达的检测,并分析其与宫颈癌患者年龄、肿瘤分化程度和浸润的关系。结果：48例宫颈癌中全部ANG蛋白免疫染色为阳性,分布部位在细胞浆和细胞核,细胞类型以癌细胞、间质成纤维细胞和血管内皮细胞表达为主。高分化组与低分化组之间（P=0.0032）、中分化组与低分化组之间ANG表达的差异有统计学意义（P=0.0265）,有浸润和无浸润组之间差异有显著性（P=0.0184）。结论：ANG蛋白在宫颈鳞癌中普遍表达,且表达水平与肿瘤分化程度以及浸润有关,提示ANG可能参与宫颈癌的发生发展。     

下调血管生成素基因表达对膀胱癌移植瘤生长及p－AKT、p －GSK3β、p －mTOR 表达的影响

目的：探讨下调血管生成素（ ANG ）基因表达,对膀胱癌移植瘤生长及p－AKT、p－GSK3β、p－mTOR表达的影响。方法采用ANG mRNA的特异性siRNA表达载体和无同源性的对照载体,在脂质体介导下稳定转染膀胱癌T24细胞,根据转染质粒不同分为3组：T24组为正常T24细胞,T24－NC组为转染阴性对照质粒T24细胞,T24－siANG组为转染干扰质粒T24细胞。通过筛选、鉴定后,将各组细胞皮下注射到BALB／c裸鼠中,观察肿瘤生长和肿瘤微血管密度、肺上肿瘤转移灶的变化,利用免疫组化技术检测CD31、p－AKT、p－GSK3β、p－mTOR的表达。结果与T24－NC组相比,T24－siANG组瘤体质量显著降低（P＜0.01）,抑瘤率为57.46％。 HE染色显示,T24－siANG组肿瘤微血管较T24和T24－NC组明显减少,T24－siANG组CD31、p－AKT、p－GSK3β、p－mTOR表达呈弱阳性反应。结论下调ANG基因的表达能显著抑制膀胱癌移植瘤生长,并通过降低CD31、p－AKT、p－GSK3β、p－mTOR表达抑制膀胱癌裸鼠移植瘤增殖及转移的潜能。     

间接ELISA法测定转基因小鼠血清中核糖核酸酶抑制因子的表达

[目的]建立间接ELISA法测定血清中核糖核酸酶抑制因子(ribonuclease inhibitor,RI)含量的方法,并测定在转RI基因小鼠血清中RI的表达情况。[方法]用血清样品包被酶标板,兔抗人RI抗体为一抗,辣根过氧化物酶标记的羊抗兔IgG为二抗,建立血清RI定量检测的间接ELISA法;取转染了RI基因的小鼠的血清,用上述建立的间接ELISA法测定不同时相小鼠血清中RI的表达水平。[结果]定量检测血清RI的间接ELISA法最适反应条件为一抗最佳稀释度为1∶4000,二抗的最佳稀释浓度为1∶2000。转基因小鼠第2、3周血清中RI含量较低(0.556±0.080),1个月左右达到最高值(0.836±0.113),3个月时明显下降(0.640±0.100)。[结论]间接ELISA法可以用于测定血清中的RI抗原含量。     

胃癌组织中血管生成素样蛋白2和miR-211表达水平与其预后的关系

目的 探讨胃癌组织中血管生成素样蛋白2(ANGPTL2)和miR-211表达水平与其预后的关系.方法 采用免疫组化法检测胃癌根治术治疗的62例患者胃癌组织及其相应癌旁组织中ANGPTL2和miR-211的表达水平,并分析其对胃癌患者预后的影响.结果 胃癌组织中ANGPTL2和miR-211阳性表达率分别为88.71%和83.87%,均显著高于其癌旁正常组织的3.23%和8.06%(P<0.05).62例患者随访3年的复发率、无病生存期(DFS)和3年生存率分别为45.16%、(16.62±4.78)个月和62.90%,且ANGPTL2和miR-211阳性表达患者的复发率高于阴性表达患者,DFS短于阴性表达患者,3年生存率则低于阴性表达患者(P<0.05).logistic线性回归分析结果显示,胃癌组织中ANGPTL2和miR-211阳性表达率均可明显影响其预后情况(P<0.05).结论 胃癌组织中ANGPTL2和miR-211阳性表达率较高且可影响其复发率、DFS和3年生存率等预后情况,胃癌组织中ANGPTL2和miR-211阳性表达可能作为其不良预后评估的参考指标.     

心肌内注射转染血管生长素基因改善心肌梗死大鼠心功能的实验研究

目的 观察心肌内注射转染血管生长素基因对心肌梗死大鼠梗死面积、非梗死区心肌肥厚以及心功能的影响.方法 成年雄性Wistar大鼠,随机分为假手术组、MI-rAAV-lacZ组和MI-rAAV-ANG组.后2组以直视下结扎冠脉左前降支的方法建立心肌梗死模型,然后取rAAV-lacZ或rAAV-ANG载体在梗死区周围多点注射.1周和4周后分别测量心肌梗死面积.4周后超声心动图测量左室后壁厚度和左室射血分数(LVEF),颈动脉插管测量有创左室血流动力学指标.大鼠处死后左室称重,计算左室重量与体重比值.Western blot方法检测大鼠左室心肌组织ANG蛋白表达水平.结果 左前降支结扎1周时,MI-rAAV-lacZ组和MI-rAAV-ANG组大鼠梗死面积无明显差异.4周后,MI-rAAV-lacZ组大鼠梗死面积显著大于1周时,亦显著大于MI-rAAV-ANG组4周时.与假手术组相比,MI-rAAV-lacZ组大鼠左室后壁肥厚、左室重量增加.4周后,与MI-rAAV-lacZ组相比,MI-rAAV-ANG组左室心肌ANG蛋白表达显著增加,梗死区扩展程度减轻,后壁厚度和左室重量恢复至假手术组水平.与假手术组相比,所有心肌梗死大鼠4周后均表现出显著的左室功能不全,rAAV-ANG转染显著改善心肌梗死大鼠左室功能.结论 心肌内注射方式转染rAAV-ANG载体显著上调心肌梗死大鼠左室心肌ANG蛋白表达水平,抑制梗死区扩展和非梗死区肥厚,部分抑制心功能不全的发生发展.     

Inhibition of prostate carcinoma establishment and metastatic growth in mice by an antiangiogenin monoclonal antibody

A neutralizing monoclonal antibody (MAb) 26-2F to human angiogenin, a potent inducer of neovascularization, has been shown previously to prevent or delay the appearance of angiogenin-secreting human colon, fibrosarcoma and lung tumor cell xenografts implanted subcutaneously (s.c.) into athymic mice. In an analogous model system, we report here that the antibody also prevents the establishment of PC-3 androgen-independent human prostate cancer tumors in, on average, 40% of treated mice (p < 0.0001, survivor analysis). Intriguingly, combining MAb 26-2F together with cisplatin and suramin, 2 therapeutic agents that together showed little antitumor activity in the aforementioned model, resulted in an even greater degree of protection (71% protected, p = 0.009 compared to antibody treatment alone). This protective effect persisted several weeks after cessation of treatment. Additionally, prophylactic systemic administration of MAb 26-2F dramatically reduced by 50% the formation of spontaneous regional metastasis originating from primary growth in the prostate gland of PC-3M cells, highly metastatic variants of PC-3. Protection from metastasis was still significant when treatment with MAb 26-2F was delayed until after the primary tumor was well established. The antibody is not directly cytotoxic to either cell type, both of which secrete angiogenin in vitro and when growing as tumors in vivo, but changes the pattern of vascularity in primary tumors growing orthotopically. These findings, together with the observation that angiogenin protein and mRNA are apparently overexpressed in cancerous vs. normal human prostate tissues, demonstrate that angiogenin antagonism represents a promising new approach for preventing progression and metastasis of clinical prostate cancer.     

Selective killing of B-cell hybridomas targeting proteinase 3, Wegener's autoantigen

Wegener's granulomatosis (WG) is a rare disease characterized by granulomatous lesions, small vessel vasculitis and the presence of anti-neutrophil cytoplasmic autoantibodies (C-ANCAs) in the sera of affected patients. Their main target antigen is proteinase 3 (PR3), a neutrophil and monocyte-derived neutral serine protease. Since the standard treatment of this severe autoimmune disease, with cyclophosphamide and corticosteroids, is associated with potential side-effects, the development of a more specific immunotherapeutic agent is warranted. The key role of ANCA in the pathogenesis of vasculitis and the effectiveness of anti-CD20 antibodies in patients with refractory WG points towards the importance of B cells in WG. We thus evaluated a new approach to selectively eliminate PR3-specific autoreactive B cells by targeting the B-cell receptor. For this purpose we used a bifunctional recombinant fusion protein consisting of the antigen PR3 and a toxin. The cytotoxic component of this novel fusion protein was the ribonuclease angiogenin, a human toxin with low immunogenicity. The toxin was stabilized by exchanging the catalytically relevant histidine in position 44 with glutamine to eliminate the autoproteolytic activity. PR3H44Q was fused either to the N terminus or to the C terminus of angiogenin. The recombinant proteins were expressed in 293T cells. Binding assays demonstrated the appropriate size and recognition by anti-PR3 antibodies. Using TUNEL technology, we demonstrated that these autoantigen toxins kill proteinase 3-specific B-cell hybridomas selectively by inducing apoptosis. The data indicate that autoantigen-toxins are promising tools in the treatment or co-treatment of autoimmune diseases in which the antigen is known.     

Levels of soluble angiogenin in chronic myeloid malignancies: clinical implications

Angiogenesis is critical for the clinical progression of haematopoietic malignancies and depends on angiogenic factors. Angiogenin is a powerful factor produced by neoplastic cells and host microenvironment. High levels of soluble angiogenin (sAng) correlate with a poor prognosis in patients affected by acute myeloid leukaemia and myelodysplastic syndromes, but no data are available on sAng in chronic myeloproliferative disorders (CMD). Therefore, in this study we investigated the clinical significance of the angiogenin in sera of patients with chronic myeloid leukaemia (CML) (n = 14) or essential thrombocythaemia (ET) (n = 20), and correlated them with those of soluble transforming growth factor-beta(1) (sTGF beta(1)). Enzyme-linked immunosorbent assay detected (P < 0.05) higher levels of sAng in CMD compared with healthy subjects (1026.74 +/- 464.60 pg/mL and 196.00 +/- 39.90 pg/mL, respectively). The highest levels of sAng were detected in CML patients (1349.23 +/- 549.55 pg/mL). Interestingly, CML patients who achieved haematological remission after interferon therapy showed circulating levels of angiogenin significantly (P < 0.05) decreased when compared with those at diagnosis. In ET patients, levels of angiogenin (889.34 +/- 267.66 pg/mL) and sTGF beta(1) (76.69 +/-6.08 pg/mL) were higher (P < 0.05) compared with healthy controls (57.93 +/- 19.39 pg/mL). No correlation was found between levels of sAng and levels of sTGF beta(1) or platelet count among ET patients. Our results show for the first time that elevated blood levels of angiogenin feature chronic myeloid malignancies, suggesting a role of angiogenin in the pathogenesis of these diseases.