重组hFGF-10腺病毒促进角质形成细胞增殖

Recombinant hFGF-10 adenovirus promotes proliferation of kerotinocytes

目的:构建重组人成纤维细胞生长因子10(hFGF-10)腺病毒,并观察其对角质形成细胞增殖的影响,为探索新的皮肤组织工程种子细胞的来源奠定基础。方法:用PCR方法扩增得到人成纤维细胞生长因子10(hFGF-10)基因片段,与穿梭载体pAdTrack-CMV连接,获得的重组质粒pAdTrack-CMV-hFGF-10经PmeI线性化后,通过电转法导入含腺病毒骨架质粒pAdEasy-1的大肠杆菌BJ5183中进行同源重组,获得重组腺病毒质粒pAdEasy-hFGF-10,经酶切鉴定后转染HEK-293细胞,在HEK-293细胞中包装并扩增腺病毒。用重组腺病毒感染HaCat细胞,Western blotting检测培养上清中的重组hFGF-10。MTT法检测重组腺病毒对角质形成细胞增殖的影响。结果:成功构建了含hFGF-10基因的重组腺病毒,可高效感染HaCat细胞,培养上清与hFGF-10抗体呈阳性反应。重组腺病毒对角质形成细胞的增殖具有促进作用。结论:制备出的重组腺病毒感染HaCat细胞后分泌表达hFGF-10,并可促进HaCat细胞的增殖。

AIM： To construct the recombinant adenoviral vector containing human fibroblast growth factor 10 （hFGF- 10） gene, and to study the effect of the recombinant adenovirus on the proliferation of kerotinocytes. METHODS ： HFGF - 10 gene was amplified by PCR and ligated with shuttle vector pAdTrack - CMV to get the recombinant plasmid pAdTrack - CMV - hFGF - 10, which was linearized with PmeⅠ and transferred into Escherichia coli BJ5183 containing the adenoviral bone plasmid pAdEasy - 1 for homologous recombination to obtain the recombinant adenoviral plasmid pAdEasy - hFGF - 10. The recombinant adenoviral plasmid was then transfected into HEK - 293 cell line to package and amplify the recombinant adenovirus. The expression of hFGF - 10 in HaCat cells infected with the recombinant adenovirus was detected by Western blotting. The influence of the recombinant adenovirns on the proliferation of kerotinocytes was checked by MTT. RESULTS： The recombinant adenovirns containing hFGF - 10 gene was successfully constructed, which effectively infected HaCat cells. The result of Western blotting showed that a protein in culture media of the infected HaCat ceils reacted with hFGF - 10 antibody. The recombinant adenovirns stimulated the proliferation of kerotinocytes. CONCLUSION： HaCat cells infected with the recombinant adenovirns expresses and secrets hFGF- 10 protein, which promotes the proliferation of HaCat cells.