VEGF-165/ANG-1双基因共转染血管内皮祖细胞复合多孔生物活性玻璃的生物相容性研究

Biocompatibility of porous bioactive glass-ceramic loaded with VEGF165 and ANG1 gene cotransfected EPCs

摘要:

目的  制备一种具有诱导血管再生活性的新型复合骨组织工程材料，并评价其生物相容性。方法  将血管内皮生长因子-165（VEGF-165）/血管生成素-1（ANG-1）双基因共转染的血管内皮祖细胞（EPCs）复合多孔生物活性玻璃，制成复合人工骨材料。采用Western blot、逆转录-聚合酶链反应（RT-PCR）检测已转染EPCs中VEGF-165及ANG-1的表达。噻唑蓝（MTT）细胞毒性试验、骨植入试验及扫描电镜观察细胞在材料表面黏附情况等综合评价材料的生物相容性；CD31免疫组织化学法染色观察材料中血管再生的情况。结果  Western blot检测、RT-PCR反应显示，实验组VEGF-165、ANG-1的表达较对照组升高。MTT细胞毒性试验、骨植入试验均显示，材料无毒性反应。扫描电镜显示，材料表面有较多血管内皮祖细胞黏附，并有较多伪足伸出。苏木精-伊红染色法（HE）染色显示，材料周围组织生长良好，未见明显的炎症细胞浸润，材料与周围组织交界处有大量的新骨生成。CD31免疫组织化学法染色显示，材料中有较多新生的毛细血管。结论  VEGF-
165/ANG-1双基因共转染EPCs复合多孔生物活性玻璃的骨组织工程材料生物相容性良好，具备一定的诱导血管再生活性。

Abstract:

Objective To prepare a novel bone tissue engineering material with angiogenesis activity, and evaluate the biocompatibility and safety of the composites. Methods Endothelial progenitor cells (EPCs) were cotransfected with VEGF165 and ANG1 genes and loaded into porous bioactive glass-ceramic to make the composite. The expressions of VEGF165 and ANG1 in EPCs were detected by RT-PCR and Western blot. MTT cytotoxicity test, bone implantation test and electron microscopic examination were used to comprehensively evaluate the biocompatibility of the composite. Immunohistochemical staining of CD31 was used to analyze revascularization of the composite zones. Results Both RT-PCR and Western blot showed cotransfected EPCs expressed increased VEGF165 and ANG1. The results of MTT cytotoxicity test and bone implantation test showed that the material was non-toxic. Scanning electron microscope displayed many EPCs adhered to the surface of the material, and had a lot of pseudopods. HE staining revealed tissue surrounding the material grew well without marked inflammatory cell infiltration. CD31 immunohistochemical staining indicated newly-formed capillaries into the material, demonstrating that the new tissue engineering material has good biocompatibility. Conclusions The composite of porous bioactive glass-ceramic loaded with VEGF165 and ANG1 gene cotransfected EPCs has good biocompatibility, and can improve angiogenesis.