膀胱癌细胞系中诱变型内皮抑素对Bcl-2蛋白表达影响的研究  

Effect of mutagenesis endostatin on expression of Bcl-2 protein in bladder cancer cell lines

作　　者：王长林[1] 任明华[1] 

机构地区：[1]哈尔滨医科大学附属第一医院泌尿外科,黑龙江哈尔滨150001

出　　处：《哈尔滨医科大学学报》2017年第2期104-108,共5页Journal of Harbin Medical University

基　　金：2014年哈尔滨市应用技术研究与开发项目(2014RFQGJ088)

摘　　要：目的研究诱变型内皮抑素对T-24膀胱癌细胞Bcl-2蛋白表达量的影响。方法运用定点诱变技术,将人内皮抑素25~31位氨基酸由RGIRGAD改为RGDRGD后,合成诱变型人内皮抑素(1~30位氨基酸对应核苷酸序列),连接到质粒p TYB2中,转化至大肠杆菌BL-21(DE3)中表达,几丁质亲和层析树脂一步纯化诱变型内皮抑素,葡聚糖凝胶G15去除DTT后经Tricine SDS-PAGE鉴定诱变型内皮抑素。诱变型内皮抑素作用体外培养T-24膀胱肿瘤细胞后,Western blot检测T-24细胞的Bcl-2蛋白表达量;MTT比色法测定对T-24细胞的增殖抑制情况;流式细胞技术测定T-24细胞的早期调亡情况。结果通过诱变技术及基因工程方法提取的诱变型内皮抑素其相对分子质量与理论大小一致;Western blot检测结果显示诱变型内皮抑素抑制了T-24细胞的Bcl-2蛋白表达;MTT法显示诱变型内皮抑素对T-24细胞有明显的增殖抑制作用,流式细胞仪检测到细胞凋亡增加。结论诱变型人内皮抑素(诱变型内皮抑素)能够明显抑制T-24膀胱肿瘤细胞Bcl-2的表达,抑制细胞的增殖,促进早期凋亡。Objective To investigate the influence of the mutation-type endostatin （endostatin, abbreviated ES） on expression of Bcl-2 protein in T-24 bladder cancer cells. Methods The 25 th - 31 st amino acids of human endostatin were changed from RGIRGAD to RGDRGD by using the site-directed mutagenesis technology, and the synthetic mutation type human endostatin gene（ 1 -30 amino acid corresponding to nueleotide sequence） was connected to the plasmid pTYB2 and transformed into E. coli BL-21 （DE3） competent cell to express the mutation type human endostatin. The mutation type human endostatin was purified by chitin resin affinity chromatography and identified by Tricine SDS-PAGE followed removal of DTT by Sephadex G15. The influence of mutation type human endostatin on the expression of Bcl-2 protein in the T-24 cells was detected by Western blot, the proliferation inhibition was examinated by MTr colorimetric assay and the early apoptosis of T-24 cell was detected by flow cytometry technique. Results Through mutagenesis and genetic engineering techniques, the molecular weight of the mutagenesis type human endostatin were obtained as the same as the theoretical molecular weight. The mutation type human endostatin obviously inhibited the expression of Bcl- 2 protein in T-24 cell by Western blot method and also significantly inhibited the T-24 cell proliferation which was found by MTT method. Conclusion Mutation-based human endostatin （30 peptides） can not only obviously inhibit the expression of Bcl-2 protein in T-24 cells, but also suppress the proliferation of T-24 cells and promote the early apoptosis of T-24 cells.

关 键 词：诱变型内皮抑素 RGD Bcl-2 抗肿瘤 膀胱肿瘤 

分 类 号：R737.14[医药卫生—肿瘤]