CAJ | 학술논문

目的:用hVEGF_(121)/EGFP真核质粒转染骨髓间充质干细胞,提纯其表达的hVEGF_(121)/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-N_2-hVEGF_(121)重组质粒提取质粒DNA.通过阳离子脂质体介导pEGFP-N_2-hVEGF_(121)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hVEGF_(121)/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-N_2-hVEGF_(121)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hVEGF_(121)/EGFP融合蛋白表达.MTT试验证实人脐静脉内皮细胞数量在hVEGF_(121)/EGFP融合蛋白组明显多于对照组(P<0.05).Miles实验证实hVEGF_(121)/EGFP融合蛋白增加血管壁的通透性.结论:①携带hVEGF_(121)/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hVEGF_(121)/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.
목적:용hVEGF_(121)/EGFP진핵질립전염골수간충질간세포,제순기표체적hVEGF_(121)/EGFP융합단백,병체외검측기공능.방법:용과제조전기공작구건적pEGFP-N_2-hVEGF_(121)중조질립제취질립DNA.통과양리자지질체개도pEGFP-N_2-hVEGF_(121)중조질립DNA전염체외배양적골수간충질간세포,사용형광현미경검측hVEGF_(121)/EGFP융합단백표체.용Amicon초려리심관순화융합단백.결과:형광현미경하관찰도전염pEGFP-N_2-hVEGF_(121)중조질립적골수간충질간세포,Westen blot증실전염골수간충질간세포적배양액중존재hVEGF_(121)/EGFP융합단백표체.MTT시험증실인제정맥내피세포수량재hVEGF_(121)/EGFP융합단백조명현다우대조조(P<0.05).Miles실험증실hVEGF_(121)/EGFP융합단백증가혈관벽적통투성.결론:①휴대hVEGF_(121)/EGFP융합단백표체기인적진핵표체질립,재골수간충질간세포중획득표체.②골수간충질간세포표체적hVEGF_(121)/EGFP융합단백재체외구유야생형혈관내피세포생장인자적공능.
OBJECTIVE: To purify hVEGF_(121)/EGFP fusion protein using transfected BMSCs as culture media, in addition, to detect the function of hVEGF_(121)/EGFP fusion protein in vitro.METHODS: The pEGFP-N_2-hVEGF_(121) 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-hVEGF_(121) by positive ionic liposome transfection method. Under a fluorescent microscopy, the expression of hVEGF_(121)/EGFP fusion protein was detected. The hVEGF_(121)/EGFP fusion protein was purified with Am icon ultrafiltration centrifuge tube and the expression of fusion protein was detected by Western-Blotting method.RESULTS: The BMSCs, which transfected with pEGFP-N2-hVEGF_(121), was observed under the fluorescent microscope. Western blotting confirmed that pEGFP-N_2-hVEGF_(121) 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-N_2-hVEGF_(121) fusion protein increased the permeability of the blood vessel wall.CONCLUSION: ①This study successfully confirmed the pEGFP-N_2-hVEGF_(121) recombinant plasmid, which carrying VEGF_(121)/EGFP fusion protein, can be expressed in BMSCs.②The VEGF_(121)/EGFP fusion protein have the function of wild-type VEGF in vitro.