【摘要】：細(xì)胞因子蛋白復(fù)雜脆弱的高級(jí)構(gòu)象極易因組織工程支架制備過程中的苛刻條件變性而失活,甚至引起有害的抗體反應(yīng)。目前有關(guān)組織工程支架材料的研究方法尚未實(shí)現(xiàn)以天然構(gòu)象長(zhǎng)期緩釋細(xì)胞因子蛋白。針對(duì)這個(gè)難題,本研究運(yùn)用水相-水相冷凍相分離蛋白顆粒制備法,將蛋白藥物血管內(nèi)皮生長(zhǎng)因子(VEGF_(165))保護(hù)性載入葡聚糖納米顆粒中,進(jìn)而將載有蛋白的多糖顆�；鞈矣谧鳛榫忈尰|(zhì)的聚乳酸-羥基乙酸共聚物(PLGA)中,通過高壓靜電紡絲,制得長(zhǎng)效緩釋VEGF_(165)的組織工程纖維膜。一系列物理化學(xué)表征,包括掃描電鏡(SEM)、接觸角、強(qiáng)度試驗(yàn)等,表明上述方法制得了表面形態(tài)與機(jī)械強(qiáng)度較理想的組織工程纖維。對(duì)制備過程中各個(gè)階段的蛋白進(jìn)行回收,并采用CCK-8法進(jìn)行活性檢測(cè)證明VEGF_(165)的生物活性在多糖顆粒制備階段保持在97%以上,在最終的組織工程纖維膜階段保持在87%以上,遠(yuǎn)大于w/o型乳液法制備得到的纖維(低于55%)。體外試驗(yàn)證明顆粒復(fù)合纖維能抑制蛋白突釋且達(dá)到長(zhǎng)效緩釋效果,蛋白在初始5 d內(nèi)的釋放總量不到35%,到25 d時(shí)釋放量超過92%。而w/o型乳液法制得的纖維在初始5 d內(nèi)的釋放總量超過53.12%,到25 d時(shí)釋放量為84.52%。最后將纖維膜植入下肢缺血的大鼠體內(nèi),進(jìn)行免疫組化觀察,結(jié)果表明顆粒復(fù)合PLGA纖維的再生血管數(shù)量明顯高于w/o型乳液法制備得到的纖維。
[Abstract]:The complex and fragile high conformation of cytokine proteins is easily inactivated due to the severe denaturation of tissue engineering scaffolds and even causes harmful antibody reactions. At present, the research methods of tissue engineering scaffold materials have not been achieved with the natural conformation of long-term sustained release cytokine protein. In order to solve this problem, the preparation of protein particles in aqueous phase freezing phase was studied. The protein drug vascular endothelial growth factor (VEGF_ _ (165) was protected into dextran nanoparticles. Then the polysaccharides containing protein were mixed in the poly (lactic acid-glycolic acid) copolymer (PLGA) as the sustained-release matrix, and the tissue engineering fiber membrane of long-lasting sustained release VEGF_ _ (165) was prepared by high-voltage electrostatic spinning. A series of physicochemical characterizations, including SEM (SEM), contact angle, strength test and so on, show that the surface morphology and mechanical strength of the tissue engineering fibers are ideal. The protein was recovered from each stage of the preparation and the bioactivity of VEGF_ _ (165) was determined by CCK-8 method. The bioactivity of VEGF_ _ (165) was over 97% in the preparation stage of polysaccharides and more than 87% in the final phase of tissue engineering fiber membrane. It is much larger than the fiber (less than 55%) prepared by w / o emulsion method. In vitro experiments showed that the granular composite fiber could inhibit the sudden release of protein and achieve long-term sustained release effect. The total amount of protein released within 5 days was less than 35%, and at 25 days the release amount exceeded 92%. In the first 5 days, the total release amount of the fibers obtained by the w / o emulsion method was more than 53.12, and at 25 days, the release amount was 84.52. Finally, the fibrous membrane was implanted into the rats with lower limb ischemia. The results showed that the number of regenerated vessels of granular composite PLGA fibers was significantly higher than that of the fibers prepared by w / o emulsion method.
【作者單位】： 上海交通大學(xué)藥學(xué)院;
【分類號(hào)】：R943

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