内皮抑制素蛋白质的制备及生物学活性测定(英文)

The preparation of endostatin protein and the measurement of its biologic activity

目的:探讨内皮抑制素蛋白质的制备及其对血管内皮细胞的生物学活性作用。方法:通过限制性内切酶酶切反应、聚合酶链式反应、DNA测序及用NCBIBLAST软件与基因库序列比较的方法进行鉴定,鉴定后在大肠杆菌中扩增,用质粒纯化试剂盒抽提纯化;将纯化的pBlast-hEn-dostatin转染人成纤维细胞,用超滤和亲和层析法初步提纯转基因成纤维细胞产生的内皮抑制素蛋白,用Rt-PCR、Western-Blot和免疫组化检测内皮抑制素的表达,用MTT法检测其对人脐静脉内皮细胞的抑制作用。结果:实验证实质粒pBlast-hEndostatin含有人内皮抑制素基因。转染的成纤维细胞可以产生内皮抑制素蛋白,内皮抑制素蛋白质对人脐静脉内皮细胞作用48h2.5,5,10,20,40,80mg/L组抑制率分别为8.5%,13.1%,27.7%,38.1%,56.7%,63.8%,经曲线拟合后,内皮抑制素作用于人脐静脉内皮细胞48hIC50值为34.5mg/L。而内皮抑制素对人成纤维细胞则没有明显抑制作用。结论:转内皮抑制素基因的人成纤维细胞可以表达内皮抑制素蛋白,表达的内皮抑制素蛋白对人脐静脉内皮细胞有生长抑制作用,而对人成纤维细胞没有生长抑制作用。

AIM： To investigate the preparation of endostatin pro- tein and its biologic activity on vascular endothelial cell. METHODS： pBlast-hEndostatin and pBlast-Mcs were identified by digesting with Nhe Ⅰ and Sal Ⅰ, by PCR reaction, by sequencing, and by Alignments of PCR products with gene bank using NCBIBLAST software. The identified pBlast-hEndostatin as well as pBlast-Mcs were then purified with QIAGEN Endofree plasmid maxi kit. The purified plasmids transfected human fibroblasts. The expression of endostatin was detected by RT-PCR, Western-Blot and immunohistochemistry. The endostatin protein produced by transfected fibroblasts was purified by ultrafiltration and affinity chromatography. The inhibitory action of endostatin on human umbilical vein endotheli- um was measured by MTT assay. RESULTS： pBlast-hEndostatin was found to contain human endostatin gene. Endostatin protein was produced by transfected fibroblasts. The inhibitory ratio of 2.5,5,10,20,40,80mg/L endostatin on human umbilical vein endothelium for 48h were 8.5%,13.1%,27.7%,38.1%, 56.7%,63.8% respectively. IC50 value was 34.5mg/L. No inhibition action was found on fibroblasts. CONCLUSIONS： Endostatin protein can be produced by the transfected fibroblasts has inhibitory action on human um and has no inhibition action The produced endostatin umbilical vein endothelion fibroblasts.