基于iRGD肽的融合蛋白的构建及其靶向抗肿瘤作用的研究

发布时间：2018-03-28 19:04

本文选题：KLA　切入点：iRGD　出处：《南京医科大学》2016年博士论文

【摘要】：第一部分KLA-iRGD融合蛋白的构建及穿透性、靶向抗肿瘤作用的研究[目的]研究KLA-iRGD融合蛋白对胃癌细胞凋亡的影响,体内治疗胃癌MKN45皮下瘤模型,观察其抑瘤活性以及对肿瘤组织的靶向穿透作用。[方法]运用MTT和流式细胞仪检测KLA-iRGD融合蛋白对胃癌细胞株凋亡的影响;建立胃癌细胞株mKN45皮下瘤模型,腹腔注射KLA-iRGD融合蛋白治疗;免疫荧光方法检测融合蛋白在肿瘤组织的分布及肿瘤组织细胞凋亡情况;检测其他器官细胞凋亡情况。[结果]KLA-iRGD融合蛋白对胃癌细胞呈现明显的促凋亡作用;在胃癌细胞MKN45皮下瘤模型中,KLA-iRGD融合蛋白经尾静脉注射后呈现明显的靶向性及穿透性,同时表现出明显的抗肿瘤作用,KLA-iRGD融合蛋白治疗组与对照组相比肿瘤直径存在显著性差异。[结论]KLA-iRGD融合蛋白对胃癌MKN45肿瘤细胞具有明显的促凋亡作用,且其在体内也有明显靶向抗肿瘤作用,体内可抑制肿瘤生长,且对其他器官没有明显影响。第二部分sTRAIL-iRGD融合蛋白的表达纯化及靶向抗肿瘤作用的研究[目的]利用原核表达系统表达纯化sTRAIL-iRGD融合蛋白,研究其对胃癌细胞凋亡的影响,体内治疗胃癌MKN45皮下瘤模型,观察其抑瘤活性以及对肿瘤组织的靶向穿透作用。[方法]使用原核表达技术表达纯化蛋白;使用考马斯亮蓝染色以及Western Blot法测定相应蛋白的表达量;使用流式细胞仪以及MTT法测定相应蛋白作用于胃癌细胞株凋亡的影响;建立胃癌细胞株MKN45皮下瘤模型,腹腔注射sTRAIL-iRGD融合蛋白治疗;使用免疫荧光方法测定蛋白在肿瘤组织内的分布情况以及肿瘤组织的凋亡。[结果]sTRAIL-iRGD融合蛋白在细胞内、三维细胞培养模型及肿瘤组织内呈现明显的抗肿瘤作用;在胃癌细胞MKN45皮下瘤模型中,sTRAIL-iRGD融合蛋白经尾静脉注射后呈现明显的靶向性及穿透性,同时表现出明显的抗肿瘤作用,sTRAIL-iRGD融合蛋白治疗组与对照组相比肿瘤直径存在显著性差异。[结论]融合蛋白sTRAIL-iRGD可以通过原核表达系统来表达纯化,融合蛋白sTRAIL-iRGD可明显促进胃癌细胞的凋亡,并且其在机体内具有明显的靶向作用,其可有效抑制肿瘤细胞的生长,且不作用于其他组织器官。
[Abstract]:Part one: construction and penetrability of KLA-iRGD fusion protein and its antitumor effect. [objective] to study the effect of KLA-iRGD fusion protein on apoptosis of gastric cancer cells, and to treat MKN45 subcutaneous tumor model of gastric cancer in vivo. [methods] the effect of KLA-iRGD fusion protein on apoptosis of gastric cancer cell line was detected by MTT and flow cytometry, and the subcutaneous tumor model of gastric cancer cell line mKN45 was established. KLA-iRGD fusion protein was injected intraperitoneally and the distribution of fusion protein in tumor tissue and apoptosis of tumor tissue were detected by immunofluorescence. The apoptosis of other organs was detected. [results] KLA-iRGD fusion protein could promote apoptosis in gastric cancer cells, and KLA-iRGD fusion protein showed obvious targeting and penetration in MKN45 subcutaneous tumor model. There was significant difference in tumor diameter between the KLA-iRGD fusion protein group and the control group. [conclusion] KLA-iRGD fusion protein has obvious apoptotic effect on gastric cancer MKN45 tumor cells. And it also has obvious anti-tumor effect in vivo, which can inhibit tumor growth in vivo. The second part was the expression and purification of sTRAIL-iRGD fusion protein and its targeted antitumor effect. [objective] to study the effect of sTRAIL-iRGD fusion protein on apoptosis of gastric cancer cells by using prokaryotic expression system. The MKN45 subcutaneous tumor model of gastric cancer was treated in vivo to observe its inhibitory activity and its targeted penetration to tumor tissue. [methods] the purified protein was expressed by prokaryotic expression technique. Coomassie brilliant blue staining and Western Blot method were used to determine the expression of corresponding proteins; flow cytometry and MTT methods were used to determine the effect of corresponding proteins on apoptosis of gastric cancer cell line. Intraperitoneal injection of sTRAIL-iRGD fusion protein was used to determine the distribution of sTRAIL-iRGD fusion protein in tumor tissue and apoptosis of tumor tissue. [results] sTRAIL-iRGD fusion protein was expressed in cells. Three-dimensional cell culture model and tumor tissue showed obvious anti-tumor effect, in gastric cancer cell MKN45 subcutaneous tumor model, the fusion protein of STRAIL-iRGD showed obvious targeting and penetration after injection through tail vein. There was significant difference in tumor diameter between the treatment group and the control group. [conclusion] the fusion protein sTRAIL-iRGD can be expressed and purified by prokaryotic expression system. The fusion protein sTRAIL-iRGD can significantly promote the apoptosis of gastric cancer cells, and it has obvious targeting effect in the body, it can effectively inhibit the growth of tumor cells, and does not affect other tissues and organs.
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.2

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