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细胞因子蛋白复杂脆弱的高级构象极易因组织工程支架制备过程中的苛刻条件变性而失活,甚至引起有害的抗体反应。目前有关组织工程支架材料的研究方法尚未实现以天然构象长期缓释细胞因子蛋白。针对这个难题,本研究运用水相-水相冷冻相分离蛋白颗粒制备法,将蛋白药物血管内皮生长因子(VEGF_(165))保护性载入葡聚糖纳米颗粒中,进而将载有蛋白的多糖颗粒混悬于作为缓释基质的聚乳酸-羟基乙酸共聚物(PLGA)中,通过高压静电纺丝,制得长效缓释VEGF_(165)的组织工程纤维膜。一系列物理化学表征,包括扫描电镜(SEM)、接触角、强度试验等,表明上述方法制得了表面形态与机械强度较理想的组织工程纤维。对制备过程中各个阶段的蛋白进行回收,并采用CCK-8法进行活性检测证明VEGF_(165)的生物活性在多糖颗粒制备阶段保持在97%以上,在最终的组织工程纤维膜阶段保持在87%以上,远大于w/o型乳液法制备得到的纤维(低于55%)。体外试验证明颗粒复合纤维能抑制蛋白突释且达到长效缓释效果,蛋白在初始5 d内的释放总量不到35%,到25 d时释放量超过92%。而w/o型乳液法制得的纤维在初始5 d内的释放总量超过53.12%,到25 d时释放量为84.52%。最后将纤维膜植入下肢缺血的大鼠体内,进行免疫组化观察,结果表明颗粒复合PLGA纤维的再生血管数量明显高于w/o型乳液法制备得到的纤维。
The more complex and fragile high-level conformations of cytokines are easily inactivated by harsh conditions during tissue engineering scaffold preparation and even cause unwanted antibody reactions. Current research methods for tissue engineering scaffold materials have not yet achieved long-term sustained release of cytokine proteins in their native conformation. In response to this problem, the present study used aqueous phase-water phase freeze-phase protein particle preparation method, the protein drug vascular endothelial growth factor (VEGF_ (165)) protective loading of dextran nanoparticles, which will carry protein The polysaccharide particles were suspended in polylactide-glycolic acid (PLGA) as a sustained-release matrix and electrospun by high-pressure electrospinning to obtain a tissue engineering fiber membrane with sustained release of VEGF_ (165). A series of physical and chemical characterization, including scanning electron microscopy (SEM), contact angle, strength test and so on, showed that the above method prepared surface morphology and mechanical strength of the ideal tissue engineering fibers. The activity of VEGF_ (165) was tested by CCK-8 method. The biological activity of VEGF_ (165) was maintained above 97% during the preparation of polysaccharide granules and kept at 87 during the final tissue engineering fiber membrane phase %, Far greater than the w / o emulsion prepared fibers (less than 55%). In vitro experiments showed that the granule composite fiber can inhibit protein burst release and achieve long-term sustained release effect, the protein release within the first 5 d less than 35% of the total amount of the release of more than 92% by 25 d. The w / o emulsion prepared fiber in the first 5 d of the total release of more than 53.12%, 25 d when the release of 84.52%. Finally, the fibrous membrane was implanted into the rat with ischemic limbs and immunohistochemistry was performed. The results showed that the number of regenerated blood vessels in PLGA fibers was significantly higher than those prepared by w / o emulsion method.