聚四氟乙烯人工血管材料作为VEGF基因载体的可行性研究

Application of polytetrafluoroethylene as VEGF gene carrier

目的：探讨聚四氟乙烯（PTFE）人工血管携栽血管内皮生长因子（VEGF）基因并转染移植部位组织细胞的可行性。方法：将pCDI-hVEGF121，质粒溶液处理过的PTFE人工血管修剪成细条，溴化乙锭（EB）标记，在紫外先下观察其吸附基因质粒的情况。将经质粒溶液处理的血管材料置于生理盐水，并于不同的时间检测溶液的吸光度，计算出溶液的VEGF基因质粒浓度，绘出体外释放曲线。将经质粒溶液处理的血管材料（基因组，n=24）和经白蛋白溶液处理的血管材料（对照组，拧=24）分别植入家兔左、右大腿肌肉，实验按不同的时间，经RT-PCR半定量检测移植血管材料周围骨骼肌中VEGF121 mRNA表达水平，Westernblot检测VEGF121蛋白表达。结杲：吸附了VEGF基因质粒的血管材料经EB标记后，在紫外光下呈蓝紫荧光。体外释放曲线显示0．5～4h基因释放较快，随后进入缓慢增长期，直至72h仍有基因的释放，但释放速率明显减慢。RT～PCR和Westernblot显示，PTFE埋植后第1天、1周、2周、4周、6周和8周的标本hVEGF121 mRNA相对于β-actinmRNA的表达量，分别为1．053±0．356、1．718±0．404、2．021±0．303、1．872±0．231、0．986±0．254和0．340±0．116；VEGF121蛋白相对表达量为0．328±0．088、1．019±0．105、2．249±0．203、2．036±0．079、1．670±0.132和0．636±0．107。结论：PTFE血管材料能携载VEGF基因质粒并进行骨骼肌的转染。

Objective： To investigate the feasibility of vascular graft material polytetrafluoroethylene （PTFE） as gene carrier of vascular endothelial growth factor （VEGF）. Methods： The PTFE strips were soaked in pCDI-hVEGF121 solution, marked with EB solution, and then observed under ultraviolet light. The PTFE strips carrying pCDI-hVEGF121 were soaked in normal saline and the value of optical density at 260 nm on different time was measured, then the controlled release curve was made. The PTFE strips carrying pCDI-hVEGF121 were implanted into the left thigh muscles of rabbits and the PTFE strips without VEGF gene were implanted into the right. The expression of VEGF121 mRNA versus β-actin mRNA in muscles around vascular graft materials was evaluated by reverse transcriptase polymerase chain reaction （RT-PCR）. The expression of VEGF121 protein in muscle was determined by Western blot method. Results： The fluorescence on PTFE strips carrying pCDI-hVEGF121 was observed under ultraviolet light. The controlled release curve demonstrated that the gene release was fast during 0.5～4 h,then slowed down afterwards,and lasted for 72 h. RT-PCR and Western blot showed VEGF121/β-actin mRNA ratios were 1. 053±0. 356,1. 718±0. 404,2. 021±0. 303,1. 872±0. 231,0. 986±0. 254,0. 340± 0. 116 and VEGF121 protein expression levels were 0. 328±0. 088,1. 019±0. 105,2. 249±0. 203, 2. 036±0. 079, 1. 670±0. 132,0. 636±0. 107 at 1,7,14,28,42,and 56 days after implantation, respectively. Conclusion： The PTFE graft can be used as carrier of VEGF gene and VEGF gene can be transferred to skeletal muscle by this method.