人血小板源性生长因子-B基因转染犬牙龈成纤维细胞的瞬时表达检测

Transient expression of exogenous human platelet-derived growth factor-B in gingival fibroblasts of dog

目的检测携带人血小板源性生长因子-B（hPDGF-B）基因的真核表达质粒在Beagle犬牙龈成纤维细胞内的表达。方法扩增和鉴定携带hPDGF-B基因的质粒EX-A0380-M03,脂质体介导的方法转染Bealge犬牙龈成纤维细胞,RT-PCR、免疫细胞化学、ELISA以及Western blot检测hPDGF-BB的表达。结果 EX-A0380-M03所携带的目的基因为hPDGF-B基因,转染牙龈成纤维细胞24 h后可观察到细胞内的绿色荧光蛋白,48 h转染效率可达18%~38%。RT-PCR、免疫细胞化学、ELISA均能检测到细胞表达hPDGF-B。Western blot证实所表达的蛋白为融合蛋白。结论携带hPDGF-B基因的真核表达载体EX-A0380-M03能被转入牙龈成纤维细胞,并成功表达一种融合蛋白。

Objective To explore transient expression of the eukaryotic expression plasmid carrying human plate-let-derived growth factor-B（hPDGF-B） in gingival fibroblasts of Beagle dog.Methods Plasmid carrying hPDGF-B（EX-A0380-M03） was amplified and identified,and then transfected into gingival fibroblasts of Beagle dog.Reverse transcription polymerase chain reaction（RT-PCR）,immunocytochemistry,enzyme-linked immunosorbent assay（ELISA） and Western bolt were choose to detect the expression of hPDGF-B.Results Target gene carried by EX-A0380-M03 was hPDGF-B.Green fluorescene protein（GFP） expressed by transfected gingival fibroblasts was observed under inverted phase contrast fluorescence microscope（IPCFM）（after 24 hours） and the transfection efficiency was 18%-38%（after 48 hours）.Serials other methods（RT-PCR,immunocytochemistry,and ELISA） mentioned above also convinced that cells expressed hPDGF-B,and the protein that was a kind of fusion protein composed of PDGF-BB and GFP was idenified by Western blot.Conclusion Eukaryotic expression plasmid carrying hPDGF-B was transfected into gingival fibroblasts successfully,and a kind of fusion protein was expressed.