Background and purpose: Tumor vasculature is increasingly recognized as a target for cancer therapy. In recent years, a fusion protein consisting of the extra cellular domain of tissue factor (truncated tissue factor, tTF) was fused to the antibody selectively binding to tumor vasculature. Antibody-truncated tissue factor(Ab-tTF) fusion protein specifically induced thrombotic occlusion of tumor vessels resulting in tumor growth retardation or regression in some types of solid tumors. However, there were still some disadvantages in the above approach. We constructed and expressed that the (RGD)_3-tTF fusion protein with peptides arginine-glycine-aspartic acid (GRGDSP, abbr. RGD)as the carrier of tTF to explore whether it bad the capability of targeting to tumor vasculature in the colonic carcinoma model. Methods: The (RGD)_3-tTF fusion gene consisting of the tTF was fused to three series-wound peptides RGD. The (RGD)_3-tTF construct was expressed in Escherichia coil BL21(DE_3). The fusion protein was purified through Nickel affinity chromatography column. The activity of inducing blood coagulation was detected by clotting assay and coagulation factor X (FX) activation assay. The specific binding to integrins α_vβ_3 was analyzed by indirect enzyme linked immunosorbent assay (ELISA). All these were compared with the fusion protein RGD-tTE Colonic nude mice models were randomly divided into 3 groups (1 nude mice per group).Tumors were stained by the (RGD)_3-tTE RGD-tTF fusion protein and tTF which were labeled with Fluorescein Isothiocyanate(FITC). The location of the (RGD)_3-tTF fusion protein in the colonic carcinoma bearing nude mice tissue was analyzed by immunofluorescence assay. Results: The (RGD)_3-tTF fusion protein retained tissue factor thrombogenic activities. With increasing concentration, the clotting time was shortened correspondingly. Under the conditions of Ca~(2+), the clotting time was 9.96±0.56 min when the concentration was 6 μmol/L(P<0.01). The (RGD)_3-tTF fusion protein could activise F X above 6 μmol/L concentration, which was similar to RGD-tTF fusion (F=0.147, P>0.05). The ability of the (RGD)_3-tTF fusion protein binding specifically to integrins α_vβ_3 was stronger than that of the RGD-tTF fusion protein in the same concentration (F=164.81, P<0.01), which was apparently indicated by the A_(405nm) 1.25 and 0.95 when the concentration was 0.24 μmol/L. Immunofluorescence assay showed that the (RGD)_3-tTF fusion protein was assembling in the tumor vasculature of the colonic carcinoma bearing nude mice. Conclusion: The (RGD)_3-tTF fusion protein which retained tissue factor thrombogenic activities could bind specifically and efficiently to tumor vasculature in the colonic carcinoma bearing mice through binding to the tumor marker integrins α_vβ_3. It might be a promising foundation for further studies on the colon cancer molecular targeted therapy with tTF as an effective factor.%背景与目的:肿瘤血管作为肿瘤分子靶向治疗的靶点受到广泛的关注,近年来有报道称利用抗体(Ab)作为组织因子(TF)胞外区截短组织因子(truncated tissue factor,tTF)的载体,表达的抗体--截短组织因子(Ab-tTF)融合蛋白能够选择性结合肿瘤血管,诱发实体肿瘤组织血管栓塞,导致肿瘤衰退,但是该方法存在一些弊端.本实验旨在研究以精氨酸-甘氨酸-天冬氨酸(GRGDSP,RGD)多肽作为tTF载体所表达的(RGD)_3-tTF融合蛋白选择性结合结肠癌裸鼠模型肿瘤血管的能力.方法:用3个串联的RGD多肽与tTF合成融合基因(RGD)_3-tTF,表达于大肠埃希菌(Escherichia coli,E.coli)BL21(DE_3),用镍柱纯化融合蛋白.用RGD-tTF融合蛋白作对照,通过凝血实验和凝血因子X(FX)活化实验检测(RGD)_3-tTF融合蛋白的凝血活性,运用间接酶联免疫吸附试验(ELISA)方法分析其特异性结合肿瘤血管标志物整合素α_vβ_3的能力.结肠癌裸鼠模型分为3组(每组1只),肿瘤组织分别用异硫氰酸荧光素(FITC)标记的(RGD)_3-tTF、RGD-tTF融合蛋白、tTF进行荧光染色,免疫荧光实验分析融合蛋白在结肠癌裸鼠模型组织的定位.结果:(RGD)_3-tTF融合蛋白保留了组织因子的凝血活性,在Ca~(2+)存在时随着融合蛋白浓度的增加,凝血时间相应缩短,浓度为6 μ mol/L时,凝血时间为(9.96±0.56)min(与对照组比较,P<0.01).(RGD)_3-tTF融合蛋白的浓度在1 μmol/L以上时能有效活化FX,其活化能力与RGD-tTF相似(F=0.147,P0.05).同浓度(RGD)_3-tTF融合蛋白与整合素α_vβ_3的结合能力强于RGD-tTF(F=164.81,P<0.01),当融合蛋白浓度为0.24 μmol/L时,(RGD)_3-tTF融合蛋白和RGD-tTF融合蛋白的A_(405nm)分别为1.25和0.95.免疫荧光实验显示,(RGD)_3-tTF融合蛋白富集于结肠癌裸鼠模型的肿瘤血管.结论:(RGD)_3-tTF融合蛋白在保留组织因子凝血活性的同时通过高效、特异地结合肿瘤血管标志物整合素α_vβ_3,选择性地定位在结肠癌裸鼠模型的肿瘤血管上,为发展tTF作为效应因子的结肠癌分子靶向治疗奠定了基础.
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