摘要
目的构建人转化生长因子β3的真核表达载体pcDNA3.1(-)TGFβ3转染NIH3T3成纤维细胞的稳定表达系统,研究转基因TGFβ3对成纤维细胞生长增殖活性的影响。方法通过脂质体介导的转染技术和G418细胞筛选法,将真核表达载体pcDNA3.1(-)TGFβ3质粒导入NIH3T3成纤维细胞,应用RTPCR与Westernblot检测hTGFβ3在真核细胞中的表达。经相差显微镜形态观察及四唑蓝比色实验法(MTT)检测细胞增殖活性。结果在10株转染和经G418反复筛选的NIH3T3成纤维细胞系中,有7株hTGFβ3mRNA及其蛋白的表达明显增强,其余3株细胞相对较弱或者没有表达。TGFβ3转基因稳定表达的NIH3T3成纤维细胞系的生长增殖明显减缓(P<0.05)。结论pcDNA3.1(-)TGFβ3真核表达载体转染NIH3T3成纤维细胞的稳定表达系统构建成功,转基因hTGFβ3在体外培养条件下可抑制成纤维细胞的增殖。
Objective To construct and identify the stable expression system of NIH3T3 fibroblast with eukaryotic expression vector of human transforming growth factor β3 （pcDNA3.1 （ - ）/TGFβ3 ）o So as to investigate the proliferation of NIH3T3 fibroblasts transfected with hTGFβ3 gene stably. Methods The stable transfection of NIH3T3 fibroblasts with recombinant plasmid expressing hTGFβ3 was established by using LipofectamineTM2000 and G418 selection. The mRNA and protein expression of TGFβ3 were detected by the RT- PCR and Western blot method, respectively. Microscope and MTT were adopted to examine the proliferation of the stable expression system of fibroblasts with hTGFβ3. Results After G418 selection, RT-PCR and Western blot analysis, 7 out of 10 cell lines transfected with pcDNA3.1 （ - ）/TGFβ3 expressed with very high level of TGFβ3 ,as compared with vector control transfectants that showed no expression, and compared with the other cell lines that expressed relatively low level. The stable transfection of NIH3T3 fibroblasts growth slowed down significantly（P 〈 0.05）. Conclusion the stable expression system of NIH3T3 fibroblast with hTGFβ3 were constructed successfully . The TGFβ3 gene could inhibit the proliferation of NIH3T3 fibroblasts in vitro.
出处
《中华整形外科杂志》
CAS
CSCD
北大核心
2006年第2期109-112,共4页
Chinese Journal of Plastic Surgery
基金
广东省科技计划项目基金(2003C32711)
