人促黄体激素释放素-血管生成素重组毒素的表达及其体外抗肿瘤效应的实验  

Experimental study on the expression and anti-tumor effects of recombinant luteinizing hormone releasing hormone-angiogenin toxin in vitro

作　　者：倪志立[1] 丁秋云 张秋航[1] 曲秋懿[1] 严波[1] 秋枫[1] 

机构地区：[1]首都医科大学宣武医院耳鼻咽喉头颈外科,北京100053 [2]延庆县医院耳鼻咽喉科,北京102100

出　　处：《中国耳鼻咽喉头颈外科》2010年第2期73-76,共4页Chinese Archives of Otolaryngology-Head and Neck Surgery

摘　　要：目的构建表达人促黄体激素释放素-血管生成素(luteinizing hormone releasing hormone-angiogenin,LHRH-PⅡ-ANG)重组毒素并研究体外抗肿瘤活性。方法利用分子生物学技术构建并在大肠杆菌中表达LHRH-PⅡ-ANG重组毒素,对以包涵体形式表达的重组毒素进行了复性。利用结晶紫法检测了复性重组毒素对人喉癌细胞系Hep2和CNE-2生长的影响并计算IC50值。结果利用基因工程技术,成功构建并在大肠杆菌中表达了LHRH-PⅡ-ANG重组毒素。重组毒素以包涵体的形式表达获得了表达。用梯度透析法对变性重组毒素进行了复性。复性后的重组毒素对正常细胞系没有毒性,而对人喉癌细胞系Hep2和CNE-2具有特异性杀伤作用,IC50值分别为21.37μg/ml和28.61μg/ml。结论成功构建了LHRH-PⅡ-ANG重组毒素,复性后的重组毒素对人喉表皮样细胞癌细胞Hep2和鼻咽癌细胞CNE-2-2的生长有明显的抑制作用。OBJECTIVE To construct recombinant LHRH-PⅡ-ANG toxin and investigate its anti-tumor effect in vitro. METHODS Recombinant LHRH-PⅡ-ANG toxin was designed and expressed as inclusion body in E.coli. Inclusion body of recombinant LHRH-PⅡ-ANG toxin was resolved in 8M urea and refolded by gradient dialysis method. Cytotoxic and growth inhibition effects of refolded LHRH-PⅡ-ANG toxin on human laryngeal tumor cell line Hep2, nasopharyngeal carcinoma cell line CNE-2 and baby hamster kidney cell line BHK-21 were evaluated by inverted microscope observation and crystal violet method. RESULTS Prokaryotic expression vector pET28a/LHRH-ANG was constructed by typical genetic techniques and transformed into E. coli. strain BL21（DE3）. After IPGT induction, recombinant LHRH-PⅡ-ANG protein was expressed as inclusion body. Refolding of LHRH-PⅡ-ANG toxin was performed by gradient dialysis. The refolded LHRH-PⅡ-ANG toxin had obvious cytotoxic effect on Hep-2 and CNE-2 cells, but BHK-21 cells not. It specifically inhibited the proliferation of Hep2 and CNE-2 cells with IC50 21.37 μg/ml and 28.61 μg/ml, respectively. CONCLUSION Recombinant LHRH-PⅡ-ANG protein was expressed in E. coli and refolded successfully. It has specific growth inhibition effects on human head and neck tumor cells Hep2 and CNE-2.

关 键 词：促性腺素释放激素 血管生成素类 免疫毒素类 肿瘤 半数抑制浓度 

分 类 号：R73-3[医药卫生—肿瘤]