骨髓间充质干细胞表达的hVEGF_(121)/EGFP融合蛋白的提纯及功能鉴定  

Expression,purification and function of hVEGF_(121)/EGFP fusion protein in bone marrow-derived mesenchymal stem cells

作　　者：何力鹏[1,2] 王耀晟[1] 周仪华[1] 姜宇[2] 吴晓玲[2] 程晓曙[1] 

机构地区：[1]南昌大学第二附属医院心血管内科,江西省南昌市330006 [2]武警江西总队医院内科,江西省南昌市330010

出　　处：《中国组织工程研究与临床康复》2009年第49期9731-9734,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：江西省科技重点攻关项目(20041B0302100);国家"十一五"科技支撑计划(2008BA168B02)~~

摘　　要：目的:用hVEGF121/EGFP真核质粒转染骨髓间充质干细胞,提纯其表达的hVEGF121/EGFP融合蛋白,并体外检测其功能。方法:用课题组前期工作构建的pEGFP-N2-hVEGF121重组质粒提取质粒DNA。通过阳离子脂质体介导pEGFP-N2-hVEGF121重组质粒DNA转染体外培养的骨髓间充质干细胞,使用荧光显微镜检测hVEGF121/EGFP融合蛋白表达。用Amicon超滤离心管纯化融合蛋白。结果:荧光显微镜下观察到转染pEGFP-N2-hVEGF121重组质粒的骨髓间充质干细胞,Westen blot证实转染骨髓间充质干细胞的培养液中存在hVEGF121/EGFP融合蛋白表达。MTT试验证实人脐静脉内皮细胞数量在hVEGF121/EGFP融合蛋白组明显多于对照组(P<0.05)。Miles实验证实hVEGF121/EGFP融合蛋白增加血管壁的通透性。结论:①携带hVEGF121/EGFP融合蛋白表达基因的真核表达质粒,在骨髓间充质干细胞中获得表达。②骨髓间充质干细胞表达的hVEGF121/EGFP融合蛋白在体外具有野生型血管内皮细胞生长因子的功能。OBJECTIVE：To purify hVEGF121/EGFP fusion protein using transfected BMSCs as culture media,in addition,to detect the function of hVEGF121/EGFP fusion protein in vitro.METHODS：The pEGFP-N2-hVEGF121 recombinant plasmid,which was constructed in the preliminary work of our study group,was used to extract the plasmid DNA.BMSCs were transfected with pEGFP-N2-hVEGF121 by positive ionic liposome transfection method.Under a fluorescent microscopy,the expression of hVEGF121/EGFP fusion protein was detected.The hVEGF121/EGFP fusion protein was purified with Amicon ultrafiltration centrifuge tube and the expression of fusion protein was detected by Western-Blotting method.RESULTS：The BMSCs,which transfected with pEGFP-N2-hVEGF121,was observed under the fluorescent microscope.Western blotting confirmed that pEGFP-N2-hVEGF121 fusion protein expressed in the culture media of transfected BMCS.MTT results showed the number of human umbilical vein endothelial cells in the fusion protein team was significantly greater than that in the control group （P〈0.05）,and Miles test confirmed that pEGFP-N2-hVEGF121 fusion protein increased the permeability of the blood vessel wall.CONCLUSION：①This study successfully confirmed the pEGFP-N2-hVEGF121 recombinant plasmid,which carrying VEGF121/EGFP fusion protein,can be expressed in BMSCs.②The VEGF121/EGFP fusion protein have the function of wild-type VEGF in vitro.

关 键 词：血管内皮细胞生长因子 骨髓间充质干细胞 融合蛋白 

分 类 号：R394.2[医药卫生—医学遗传学]