Canstatin分泌增强体系的建立及其抗肺癌作用

目的:评估canstatin分泌增强体系在不同氧条件下的生物学效应,观察其对裸鼠肺癌模型的抗肿瘤作用。方法:基因重组技术构建canstatin分泌型载体,将其转染肿瘤细胞观察其在不同氧条件下的canstatin mRNA表达量,稳定转染的肺癌细胞与人脐静脉内皮细胞混合培养,观察混合培养对内皮细胞增殖、凋亡的效应。建立裸鼠肺癌移植瘤模型,将此体系转染荷瘤裸鼠,观察其对裸鼠移植瘤血管生成的作用,及肿瘤体积重量等指标,评估其抗肺癌效果。结果:成功构建canstatin缺氧增强分泌体系。缺氧条件下其转染的肺腺癌A549细胞canstatin mR-NA的表达显著增加。其稳定转染的A549细胞与人脐静脉内皮细胞HUVEC混合培养可使HUVEC生长增殖缓慢并大量凋亡,此体系转染的裸鼠移植瘤生长缓慢,瘤重、瘤体积及血管生成均显著低于对照组。(P<0.01)。结论:canstatin分泌增强体系在缺氧条件下能显著增加目的基因表达量及其生物学效应,并具有显著抑制肺癌生长和血管生成的作用。

Canstatin high level expression system and its biology effects on lung cancer

Purpose:To study the biological effects of high secreted canstatin on human umbilical vein endothelial cells (HUVEC) and tumor model. Methods:Hypoxic responsive elements (HREs) were inserted in the upper stream of canstatin cDNA of a recombinant vector we constructed in a former research. The new recombinant vector named pCMV-3HRE-CEAS-Cans was transferred into A549 cells by cationic liposome; canstatin mRNA expression in the transformed cells under oxic and anoxic condition was detected by Taqman PCR. Then we designed a co-cultured assay: HUVECs were co-cultured with recombinant vector transformed A549 cells with Transwell plates, and the proliferation and apoptosis of co-cultured HUVECs were evaluated with 3H-thymidine incorporation method, TUNEL method respectively. Finally the vector was introduced into tumor tissues of lung cancer-bearing nude mice, and the biological activity in the tumor tissues was tested by micro-vessel count (MVC). A vector of canstatin we constructed before was used as controls in above experiments.Results:The pCMV-3HRE-CEAS-Cans vector was successfully constructed and transferred into A549 cells. canstatin mRNA was detected both in the recombinant vector transformed A549 cells and the pCMV-CEAS-Cans transformed A549 cells, moreover the expression of canstatin in the new vector transformed A549 was significantly higher than that of the controls under hypoxic condition. ~(3)H-TdR intake rate of HUVECs was decreased markedly, and a large amount of them underwent apoptosis when they were co-cultured with recombinant vector transformed A549 cells. Significant differences of ~(3)H-TdR intake rate and apoptotic rate of co-cultured HUVEC were found between pCMV-3HRE-CEAS-Cans group and any of the controls (P<0.05). The results of MVC showed that microvessels in the tumors transformed with pCMV-3HRE-CEAS-Cans are much less than that of any control tumors (P<0.01).Conclusions:canstatin mRNA expressed in the pCMV-Script-Cans transformed A549 cells, the expression increased under anoxic condition. The high level expression system can powerfully inhibit proliferation and induce apoptosis in co-cultured HUVECs. Furthermore, the system has good anti-tumor effects in vivo.