酸性成纤维细胞生长因子基因真核表达载体的构建及转染肌卫星细胞

Construct eukaryotic expression vector with aFGF gene and transfect muscle satellite cells

目的探讨通过构建酸性成纤维细胞生长因子（aFGF）基因真核表达载体转染大鼠肌卫星细胞（MSCs）建立细胞工程种子细胞可行性。方法首先提取人类肌肉组织总RNA，RT-PCR法调取aFGF基因，然后用直接化学合成法合成人类白细胞介素2信号肽序列（SPS），通过基因融合得到SPS-aFGF,再通过定向克隆到真核表达载体pEGFP-N1，最终得到重组质粒PEGFP-N1-SPS-aFGF，对重组质粒做测序和酶切鉴定；差速贴壁法获取大鼠MSCs，观察细胞形态，做免疫荧光鉴定；经鉴定正确的细胞随机分为3组：目的基因组（aFGF加N1）、空载质粒组（N1）、空白对照组（blank），前两组分别加入质粒PEGFP-N1．SPS-aFGF与pEGFP-N1质粒，空白对照不加入任何质粒，均在脂质体Lipofectamine2000TMReagent介导下转染，转染后荧光显微镜统计发绿色荧光的细胞占各组细胞比例，计算转染效率；转染72h后利用实时荧光定量PCR与Westernblot检测aFGF基因在MSCs中表达情况。结果①重组质粒测序结果与GenBank公布的序列一致，酶切鉴定条带与实际大小相符。②荧光显微镜下观察到转染细胞有荧光表达，并在转染72h达到最高峰。③转染后72h，实时荧光定量PCR结果显示目的基因组表达量平均相对比值（aFGF加N1）组（1464．96）显著高于N1组（1．016）、Blank组（1．000），差异有统计学意义（P〈0．05）。Westernblot检测显示aFGF表达呈极强阳性，而N1组及Blank组aFGF表达极弱。结论成功构建aFGF基因真核表达载体并转染MSCs，aFGF在MSCs内高表达，此种方法有希望获取具备特殊生物学功能细胞。

Objective To construct human acidic fibroblast growth factor （aFGF） recombinant eu karyotic expression vector and transfect it into muscle satellite cells（MSCs） of rat, in purpose of further study the method to set up cell bank. Methods The aFGF gene was cloned from human total RNA which was obtained from human skeletal muscle tissue by RT-PCR method. Human interleukin 2 （IL-2） signal peptide sequence （SPS） was obtained by direct chemosynthesis method. Then aFGF and SPS were fused to obtain SPS-aFGF. Finally, directional cloning SPS-aFGF into pEGFP-N1, the recombinant （pEGFP-N1-SPS-aFGF） was obtained. The recombinant was confirmed by endonuclease digestion and DNA sequencing. MSCs were purified by difference-speed adherence method and were ideontified by immunofluorescence assay. The correct cells were divided into 3 groups：Experimental group（aFGF＋N1）, control group（N1）, blank group（blank）. All the groups were transfected by Lipofectamine 2000TM Reagent, and pEGFP-N1-SPS-aFGF, pEGFP-N1 were respectively added in experimental group and control group while blank group was added none plasmid. Fluorescence microscope was employed to detect transfection efficiency tendency along with time changes. The expression of target gene was detected by fluorescent quantitation PCR and Western blot. Results （ 1 ） Thesequencing of pEGFP-N1-SPS-aFGF was completely correct and the outcome of endonuclease was equal to actual ban s-ize. （2）The expression of GFP in transfected cells were observed by fluorescencemicroscope and transfection efficiency reached the peak at 72 h. （3）Real-time fluorescent quantitation PCR proved strong aFGF mRNA expression in transfected cells （the average relative expression of experimental group was 1464.95） with aFGF gene, while it was detected a little in the other groups （the average relative expression of control group was 1.016 and blank group was 1.000） （P 〈 0.05）. Western blot also proved strong expression in Experi- mental group then the other two groups. Conclusion aFGF eukaryotic expression vector was successfully constructed and transfected into MSCs. This study may be expected to obtain some specific functions cells.