摘要
目的 构建可在哺乳细胞中表达碱性成纤维细胞生长因子 (basicfibroblastgrowthfactor ,bFGF)的荧光真核表达载体 ,以便进一步转染成骨细胞 ,研究bFGF基因转移在骨组织工程学中的应用。方法 应用基因重组技术 ,将已经克隆的bFGF基因从PBR3 2 2 -bFGF载体上亚克隆到荧光真核表达载体pEGFP -C3 ,构建的重组质粒经脂质体介导转染 3T3细胞 ,3 6h后观察瞬时表达情况。结果 ①酶切、PCR和DNA序列鉴定均证实插入片段的正确性。②荧光显微镜下观察GFP的表达情况 ,观察到部分经转染的细胞发出绿色荧光 ;免疫组化检测bFGF的表达分泌情况 ,结果显示部分经转染的细胞呈阳性 (棕色颗粒 )。结论 成功地构建了bFGF荧光真核表达载体pEGFP -C3 -bFGF ,并在 3T3细胞中表达了bFGF。
Objective To construct a mammalian basic fibroblast growth factor (bFGF)recombinant vector for application of bFGF gene transfer in bone tissue engineering. Methods bFGF gene was subcloned into pEGFP-C3 vector from PBR322 vector by means of gene cloning to obtain pEGFP-C3-bFGF plasmid. 3T3 cells were transfected with pEGFP-C3-bFGF plasmid using Lipofectamine TM 2000 reagent. After 36 hours, the transient expression of GFP and bFGF was measured.Results ①Correct construction of pEGFP-C3-bFGF was identified by methods of restriction enzyme analysis, PCR amplication and nucleotide sequence determination. ②Green fluorescence was emitled from transfected cells under fluorescent microscope, immunohistochemisty showed parts of transfected cells expressed bFGF (brown staining). Conclusion The pEGFP-C3-bFGF fluorescent eukaryotic expression plasmid was constructed successfully and bFGF can be expressed in 3T3 cells.
出处
《广东医学》
CAS
CSCD
2002年第8期796-798,共3页
Guangdong Medical Journal
基金
本课题为广东省自然科学基金资助项目 (编号 :990 0 0 6)
