VEGFR-3基因转染淋巴管内皮祖细胞后可溶性VEGFR-3蛋白的分泌  

VEGFR-3 Gene Transfection for Its Soluble VEGFR-3 Release in Lymphatic Endothelial Progenitor Cells

作　　者：敖红[1] 谭玉珍[1] 王海杰[1] 国海东[1] 

机构地区：[1]复旦大学上海医学院人体解剖与组织胚胎学系,上海200032

出　　处：《中国实验动物学报》2009年第4期252-257,F0002,共7页Acta Laboratorium Animalis Scientia Sinica

基　　金：国家自然科学基金资助项目(编号:30570948);高等学校博士点专项科研基金(编号:20030246036)

摘　　要：目的研究携带BABL/c小鼠VEGFR-3(1-3Ig)基因的重组腺病毒转染淋巴管内皮祖细胞(LEPCs)后,对可溶性VEGFR-3蛋白分泌及其生物学特性的作用。方法采用巢式RT-PCR技术从BABL/c小鼠胎盘组织中扩增VEGFR-3(1-3Ig)的编码基因,并通过重组PCR技术在基因的N端加上人CD33的信号肽。将该基因片段亚克隆入腺病毒表达载体(pDC316-IRES-EGFP),重组质粒经酶切及测序验证后,与包装质粒共转染293细胞以产生重组腺病毒。将构建好的携带有小鼠VEGFR-3(1-3Ig)基因的重组腺病毒转染淋巴管内皮祖细胞,通过ELISA检测转染细胞上清液中可溶性VEGFR-3蛋白的分泌及其对VEGF-C的中和作用。结果成功构建了带有信号肽的BABL/c小鼠VEGFR-3(1-3Ig)基因的腺病毒表达质粒,并获得高滴度的携带有小鼠VEGFR-3(1-3Ig)基因的重组腺病毒,重组腺病毒转染淋巴管内皮祖细胞后,可使转染细胞分泌可溶性VEGFR-3蛋白,该蛋白具有中和VEGF-C的作用。结论成功地制备了携带BABL/c小鼠VEGFR-3(1-3Ig)基因的重组腺病毒,用该病毒转染淋巴管内皮祖细胞可使其分泌可溶性VEGFR-3,该蛋白在体外具有中和VEGF-C的作用,这为临床抑制肿瘤淋巴管新生奠定了基础。Vascular endothelial growth factor receptor 3（VEGFR-3）,a specific receptor of vascular endothelial growth factor C（VEGF-C）,is a new member of tyrosine kinase receptor family.VEGFR-3 is consisted of extracellular,transmembrane and intracellular regions.The extracellular region comprises of seven immunoglobin-like domains,among which the first three domains（1-3Ig） play especially important roles in VEGF-C/VEGFR-3 signaling pathway,because these domains are responsible for the binding between VEGFR-3 and VEGF-C.Lymphatic endothelial progenitor cells（LEPCs） are mainly localized to the bone marrow,but can be released from bone marrow into diseased tissues in case of inflammation,ischemia or tumour,etc,and differentiated into lymphatic endothelial cells（LEC） to participate in the lymphangiogenesis in tumour tissues.As VEGF-C/VEGFR-3 signaling pathway is critical for the differentiation of LEPCs into LEC in tumour tissue,blockage of this signaling pathway will therefore be useful for inhibiting lymphangiogenesis and for the anti-tumour treatment.It is suggested that soluble VEGFR-3 can interfere with the binding between the VEGF-C and membrane-bound VEGFR-3,and thus can block VEGF-C/VEGFR-3 signaling pathway.In this study,we investigate the secretion of soluble VEGFR-3 proteins in LEPCs after transfected with recombinant adenovirus containing VEGFR-3（1-3Ig） gene of BABL/c mouse and the biological activity of the secreted protein.The gene encoding VEGFR-3（1-3Ig） was obtained through nested RT-PCR from BABL/c mouse placenta,and human CD33 signal peptide was added to the N-terminal of the PCR product through recombinant PCR technique.Then the gene fragment was cloned into the adenovirus shuttle plasmid pDC316-IRES-EGFP.After confirmed by restriction enzyme digestion and DNA sequencing,the recombinant adenovirus vector carrying VEGFR-3（1-3Ig） was transfected into 293-cells together with the packing vector to produce the recombinant adenovirus.The recombinant adenovirus was used to transfect L

关 键 词：BABL/C小鼠 VEGFR-3 腺病毒载体 转染 淋巴管内皮祖细胞 

分 类 号：R332[医药卫生—人体生理学]