摘要
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.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2007年第2期213-216,共4页
Chinese Journal of Pathophysiology
基金
国家"863"计划资助项目(No.2002AA2Z3318No.2004AA2Z3C60)
国家自然科学基金重点资助项目(No.303230350)