摘要
目的:通过构建人血管能抑制素(Canstatin)真核表达载体,转染裸鼠肿瘤局部,探索其对肺癌治疗作用。方法: RT-PCR法获取Canstatin cDNA全长,定向克隆法构建Canstatin基因表达载体pCMV-Script-Cans。荧光定量PCR法检测转染 细胞Canstatin mRNA的表达。台盼蓝拒染法、3H-TdR掺入法检测细胞生长增殖,TUNEL法检测细胞凋亡。基因枪转染重组 载体到荷瘤裸鼠肿瘤局部,CD31单克隆抗体微血管记数检测抗血管生成效应。结果:成功构建pCMV-Script-Cans重组载体, 并在转染的细胞株中检测到Canstatin mRNA的表达。人脐静脉内皮HUV-EC-C细胞株pCMV-Script-Cans质粒转染组比空载 体组3H-FdR掺入量明显减低(P<0.01),细胞凋亡率明显增加(P<0.01),重组载体转染肿瘤生长缓慢,微血管数显著低于对 照组(P<0.01)。结论:pCMV-Script-Cans重组载体能在转染的哺乳动物细胞中表达Canstatin,并抑制内皮细胞增殖,而且有 很好的抗肺癌血管生成作用。
Objective: To construct a mammal expression system of human canstatin and study its anti-tumor effects on lung cancer. Methods: Canstatin cDNA was acquired by RT-PCR, and cloned into a mammal exprssion vector named pC MV-Script. Canstatin expression was detected by Real-time PCR. The proliferation, apotosis of the cells transfected with recombinant canstatin vector were measured by trypan blue exclusive assay, 3H-thymidine incorporation and TUNEL method respectively. Then the recombinant vector encoding canstatin cDNAs was transferred into tumors of cancer-bearing nude mice with electroporator in vivo, and micro-vessel count was proceeded of each tumor by anti-CD31 antibody immunohisto chemical staining. Results: The recombinant vector pCMV-Script-Cans was successfully constructed , and the canstatin mRNA was detected in both of the transformed HUVE and A549 cells. The 3H-TdR intake rate in pCMV-Script-Cans transformed HUVE cells is significant lower than that of the naked plasmid transformed cells (P < 0.001) , while the apotosis rate of them is significant higher than that of the control cells (P < 0. 001). The micro-vessels in the recombinant vector transformed tumors were significant lower than that of the control group. Conclusions: Canstatin only inhibit cell proliferation and induce apotosis in endothelial cell, and it also has a good anti-tumor effect in vivo.
出处
《中国肿瘤生物治疗杂志》
CAS
CSCD
2004年第4期257-262,共6页
Chinese Journal of Cancer Biotherapy
