分泌人内皮抑素的微囊化基因工程细胞系的构建  

Microencapsulation of a genetically engineered cell line secreting human endostatin

作　　者：蔡善君[1] 詹文芳[1] 刘锐[1] 谢兵[1] 李红[1] 宿罡[1] 

机构地区：[1]遵义医学院附属医院眼科,563003

出　　处：《中华眼底病杂志》2010年第4期367-371,共5页Chinese Journal of Ocular Fundus Diseases

基　　金：贵州省科技厅自然基金（黔科合J字[2005]2061号）

摘　　要：目的 建立能稳定分泌人内皮抑素（hES）的基因工程细胞系，观察内皮抑素（ES）蛋白和hES的表达。方法以hES重组质粒pcDNA3．0（pcDNA3-Endo）为模板，通过聚合酶链反应（PCR）扩增获得hES基因片段，且在基因前加信号肽序列，将其定向插入真核表达载体绿色荧光蛋白（pEGFP—N1）质粒中，获得重组质粒pEGFP—N1-ES；利用阳离子脂质体介导将其转染到人胚胎肾细胞（HeK-293）细胞中，G418筛选后得到阳性克隆hES／293，采用蛋白免疫印迹（Westernblot）法检测转染细胞上清中ES蛋白的表达；用海澡酸钠壳聚糖（ACA）微囊包裹hES／293细胞，分别收集培养3、7、21、35d的AcA微囊化hES／293细胞的培养上清液，Westernblot法检测包裹后培养液上清中hES的表达。结果重组质粒pEGFP-N1-ES经限制性核对内切酶HindⅢ和限制性核酸内切酶BamHI双酶切得到4700碱基对（bp）和600bp2条带；PCR扩增出600bp条带；测序结果与NCBI上序列比对软件（BLAST）比对，同源性达到100％。pEGFP—N1-ES转染HeK-293细胞，经G418筛选后获得阳性克隆，选取筛选的10株单克隆细胞培养上清液进行Westernblot分析，在相对分子质量为20×10。处出现蛋白条带。在ACA微囊内hES／293细胞随着培养时间的延长，细胞团逐渐长大，充满整个囊内空间。培养3、7、21、35d时，在相对分子质量为20×10^2处出现蛋白条带。结论重组pEGFP—N1-ES真核表达载体构建正确，转染HeK-293细胞后可有效的表达hES蛋白，并能分泌到细胞外；微囊化hES／293细胞产生的ES蛋白可以自由扩散出微囊膜外，并呈持续性表达。Objective To microencapsulate a genetically engineered cell line which stably secrete human endostatin （hES）. Methods Endostatin gene fragment was amplified from plasmid pcDNA3-Endo by polymerase chain reaction, and inserted into mammalian eukaryotic expression vector pEGFP-N1, resulting into recombinant plasmid pEGFP-N1-ES. Hek-293 cells were transfected with pEGFP-N1-ES via cationic liposome and selected by G418, and were measured by Western blot for endostatin protein expression. The hES/293 cells were further entrapped by alginate-chitosan-alginate （ACA） microcapsules, and the expression of endostatin in the supernatant of cultured hES/293 cell microcapsules was examined by western blot at different time points. Results Recombinant plasmid pEGFP-Nl-endostatin was digested by HindffI and BamHI, and resulted into 2 DNA fragments of 7 kb and 600 bp. The sequence of the 600 bp fragment was identical to human endostatin. Western blot of the supernatant of cultured hES/293 cells or hES/293 cell mieroeapsules detected a positive band with the relative molecular mass of 20 × 10^3. Conclusion The hES protein was expressed in HeK-293 transfected with pEGFP-N1-endostatin, and secreted to the culture medium, and can freely diffused outside the micro-capsule.

关 键 词：内皮抑素类/生理学 微囊藻属/细胞学 细胞系 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]