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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。
Objective: To investigate the preparation of endostatin protein and its biological activity on vascular endothelial cells. Methods: The restriction endonuclease digestion reaction, polymerase chain reaction, DNA sequencing and NCBIBLAST software were used to identify and compare with the gene bank sequence. After identification, it was amplified in E. coli and extracted with plasmid purification kit The purified pBlast-hEn-dostatin was transfected into human fibroblasts. Endostatin protein produced by transgenic fibroblasts was purified by ultrafiltration and affinity chromatography. The expression of endostatin protein was detected by Rt-PCR, Western-Blot and immunohistochemistry Endostatin expression was detected by MTT assay of human umbilical vein endothelial cells. Results: The experiment confirmed that plasmid pBlast-hEndostatin contains human endostatin gene. Transfection of fibroblasts can produce endostatin protein, endostatin protein on human umbilical vein endothelial cells 48h2.5,5,10,20,40,80 mg / L group were 8.5% inhibition rate, 13.1%, respectively, 27.7%, 38.1%, 56.7% and 63.8%, respectively. After the curve fitting, the 48 hIC50 value of endostatin on human umbilical vein endothelial cells was 34.5 mg / L. Endostatin on human fibroblasts was no significant inhibitory effect. CONCLUSION: Endostatin gene-transfected human fibroblasts can express endostatin protein. The expressed endostatin protein can inhibit the growth of human umbilical vein endothelial cells and inhibit the growth of human fibroblasts.