bFGF基因修饰雪旺细胞表达的实验研究

Exogenous bFGF Gene Transfection and Expression of Its mRNA and Protein in Schwann Cells of Rats

目的利用基因工程技术将外源bFGF基因以质粒pcDNA3.1为载体导入大鼠雪旺细胞,观察外源bFGF基因在雪旺细胞中的表达水平。方法通过预变性大鼠坐骨神经、混合酶消化法获取高纯度的雪旺细胞;将真核表达质粒pcDNA3.1及外源bFGF基因片段双酶切,酶切片段回收后重组bFGF-pcDNA3.1质粒;通过脂质体转染法将pcDNA3.1-bFGF质粒导入雪旺细胞,用ELISA及RT-PCR方法分别从蛋白质水平及mRNA水平检测bFGF的表达情况。结果经外源bFGF基因修饰的大鼠雪旺细胞,bFGF的表达水平极大提高(H=126.835,v=2,P=0.000),并且相对持续、稳定。结论bFGF基因修饰的雪旺细胞能持续稳定高水平地表达分泌bFGF,将bFGF基因与雪旺细胞移植载体相结合治疗脊髓损伤具有独特的优势,为下一步bFGF基因修饰细胞治疗大鼠脊髓损伤的实验研究奠定了基础。

Objective To clone basic fibroblast growth factor （bFGF） gene, construct a recombinant eukaryotic expression plasmid pcDNA3.1 containing exogenous bFGF cDNA for studying the expression level of bFGF in Schwann cell （SC） of rat. Methods The highly purified Schwann cells were obtained by mixed enzyme digestion from lesioned sciatic nerves of rats. The bFGF cDNA was obtained from bFGF-pET3c with PCR and inserted into EcoRI and XhoI site of the plasmid pcDNA3.1, bFGF-pcDNA3.1 was transfected into SC by liposome infection method and the bFGF expression level was tested by RT-PCR and bFGF ELISA kit.Results The bFGF expression level of bFGF-pcDNA3.1 transfected SC was high （H=126.835,v=2,P=0.000）, moreover, Schwann cells modified by bFGF- pcDNA3.1 can express and excrete bFGF stably.Conclusion It is of unique advantages to connect Schwann cell and the recombinant eukaryon expres- sion plasmid bFGF-pcDNA3.1,which eould support and lead to the neuro-axon regeneration. Therefore, it offers the significant conditions for the empirical study about the damaged spinal neuron＇s rehabilitation and regeneration of rat spinal injury by transplanting Schwann cells modified by bFGF- peDNA3.1.