摘要
目的:用基因工程方法改造成纤维细胞,筛选出能够持续分泌人血管内皮细胞生长因子(hVEGF165)的细胞株,为难于愈合创面(如糖尿病性慢性溃疡)的治疗提供有效方法。方法:将含有hVEGF165的穿梭质粒pCDNA3-hVEGF165导入NIH3T3细胞,使用G-418根据有限稀释法筛选出7个单克隆细胞株。挑选出整合有hVEGF165基因且表达最高的一个细胞克隆在DNA、mRNA和蛋白质水平进行鉴定,同时使用四甲基偶氮唑蓝比色法(MTT)、鸡胚实验检测表达产物的生物学活性。结果:PCR和Southern印迹证明hVEGF165基因已整合入NIH-3T3细胞基因组,RT-PCR和Northern印迹则表明该细胞株能够表达hVEGF165-mRNA。用hVEGF165-ELISA法证实在蛋白质水平该细胞株能够持续表达hVEGF165 3个月以上,并始终保持非常高的水平。MTT实验显示其具备较强的促进内皮细胞增殖的功能,而鸡胚实验则表明该工程细胞有很强的促进胚胎血管生成的作用。结论:成功地制备了hVEGF165单克隆工程细胞,为创伤愈合的基因治疗提供实验准备。
Objective To prepare and identify monoclonal engineering cells that were integrated with hVEGF165 gene. Methods pCDNA3-VEGF was transfected into NIH3T3 cells mediated by cationic liposome, hVEGF165-integrated cell clones were screened byusing G-418 and verified by PCR and Southern blot. Meanwhile its expression of hVEGF165 was analyzed by the methods of RT-PCR, Northern blot and hVEGF165-ELISA and its biological activities were measured by MTY and CAM assay for angiogenesis in chick embryo. Results Seven hVEGF165-integrated cell clones were screened and the cell clone with the highest expression level of hVEGF165 was selected. Such cell clone was proved to integrate with hVEGF165 gene and express hVEGF165-mRNA. The test of hVEGF165-ELISA and biological activities showed that the cell clone expressed hVEGF165 in high level for over 3 months and the hVEGF165 in the supernatant could enhance the viability of vascular endothelial cells and accelerate the angiogenesis in chick embryo. Conclusions hVEGF165-integrated monoclonal engineering cell has been prepared successfully and can be used in future experiments in gene therapy for wounds.
出处
《外科理论与实践》
2006年第1期49-53,共5页
Journal of Surgery Concepts & Practice
基金
上海市教委重点项目(转化02)
上海教委第四期重点学科(ZDXK2001)资助项目
