载bFGF基因腺相关病毒与骨支架材料复合的电镜观察

Electron microscope observations of different scaffold materials loading with rAAV2-bFGF

目的:探讨载bFGF基因的腺相关病毒(rAAV2-bFGF)与不同骨支架材料的复合情况,为颅颌面骨基因治疗提供实验依据。方法:验证腺相关病毒是否携带目的基因。在真空情况下将载bFGF基因的腺相关病毒分别与聚D,L-乳酸/羟基磷灰石(PDLLA/HA)和无机牛骨(Bio-Oss)两种支架材料充分混匀,4℃过夜,在透射电镜和扫描电镜下观察载bFGF基因的腺相关病毒与支架材料的复合情况。结果:物理滴度为2.5×1011v.g./ml的rAAV2-bFGFPCR产物鉴定结果正确,能够满足进一步实验的要求。扫描电镜观察载bFGF基因的腺相关病毒在PDLLA/HA和Bio-Oss两种材料表面均有分布、黏附。透射电镜可见载bFGF基因的腺相关病毒渗透入PDLLA/HA及Bio-Oss两种材料中。结论:载bFGF基因的腺相关病毒在PDLLA/HA和Bio-Oss中均有黏附及渗透,研究PDLLA/HA和Bio-Oss材料的自身特点,综合分析认为PDLLA/HA和Bio-Oss均可作为下一步基因治疗中病毒载体的支架材料。

Objective： To evaluate the effect of different scaffold materials loading with the recombinant adeno-associated virus-mediated basic fibroblast growth factor （rAAV2-bFGF）. This may provide the basis for further craniofacial gene therapy. Method： .To identify the expression of the bFGF gene in the recombinant virus. The virus was misced bene with PDLLA / HA or Bio-Oss in vacuum, and cultured overnight in 4℃. Then the compounds were examined the combination with transmission electron microscope and scanning electron microscope. Result： The bFGF cDNA sequence was construced successfully into the recombinant virus. The physical titer of the virus was 2.5× 10^11 v.g. / mhwhich is sufficient for further experiment. In TEM and SEM observation, both PDLLA / HA and Bio-Oss were combined with rAAV2-bFGF significantly. Conclusion：This study demonstrate that both PDLLA / HA and Bio-Oss can be ulitized as scaffold materials for further craniofacial gene therapy.