Canstatin基因表达载体的构建及其生物学效应研究

目的　构建可表达人血管生成抑制素 (canstatin)蛋白的真核表达载体 ,探索canstatin对人脐静脉内皮细胞系(HUVEC)和人肺腺癌A5 49细胞株的生物学效应。方法　RT PCR法从胎儿肝组织中获取canstatincDNA全长 ,并将其克隆到真核蛋白表达载体pCMV Script上。阳离子脂质体介导该重组载体转染HUVE细胞系、A5 49细胞株。RT PCR法检测其对canstatinmRNA的表达。台盼蓝拒染法活细胞记数 ,3 H TdR掺入法检测细胞增殖 ,TUNEL法检测细胞凋亡 ,流式细胞术检测细胞周期。结果　成功构建pCMV Script Cans真核表达重组载体 ,并在转染该表达载体的A5 49及HUV EC C细胞株中均检测到canstatinmRNA的表达。HUV EC C细胞株pCMV Script Cans载体转染组比空载体组 3 H TdR掺入量明显减低(P <0 0 1) ,细胞凋亡率明显增加 (P <0 0 1) ,A5 49细胞株转染组与空载体组 3 H TdR掺入量无显著差异 (P >0 0 5 ) ,细胞凋亡率无显著差别 (P >0 0 5 )。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 ,诱导内皮细胞凋亡 ,但对肿瘤细胞没有直接抑制作用。

Construction of canstatin vector and research on its biological effects

Objective To construct an expression vector (pCMV-cans) containing canstatin cDNA and to study the biological effects of canstatin on human endothelial cells and tumor cells. Methods Canstatin full-length cDNA, acquired from adult hepatocytes by RT-PCR method, was cloned into eukaryotic expression vector pCMV-Script. The recombinant vector pCMC-cans was transfected into human umbilical vein cells (HUVECs) and human lung adeno-carcinoma cells A549 by cationic liposome. Canstatin mRNA expression in the transfected cells was detected by RT-PCR method. The proliferation, apoptosis, and cell cycle of the two kinds of cells were measured by 3H-thymidine incorporation method, TUNEL method, and flow cytometry, respectively. Results The pCMV-cans vector was successfully constructed and transfected into HUVECs and A549 cells. The canstatin mRNA was detected in both of the transfected cells. The 3H-TdR incorporation in pCMV-cans transfected HUVECs was significantly lower than that in the pCMV-Script transfected cells (P<0.01), while the apoptosis rate was significantly higher than that of the control cells (P<0.01). No significant difference in 3H-TdR incorporation and apoptosis rate was found between pCMV-cans and pCMV-Script transfected A549 cells (P>0.05). Conclusion Expression of canstatin mRNA is found in both A549 and HUVECs, but pCMV-cans transfection can only inhibit cell proliferation and induce apotosis in endothelial cells.