【作者】 刘美洲；

【导师】 彭健；

【作者基本信息】 中南大学 ， 外科学， 2007， 硕士

【摘要】 目的:本研究的目的旨在制备纳米载体,将柔红霉素纳米化,建立简便、易行的能够模拟在体血脑屏障的体外细胞模型。为柔红霉素纳米粒的临床应用提供实验依据。方法:①首先选择聚氰基丙烯酸酯为载体材料,用乳化聚合法制备柔红霉素聚氰基丙烯酸正丁酯纳米粒DAM-PBCA-NP,通过单因素试验初选、均匀设计试验、并以柔红霉素纳米粒的载药量、包封率、平均球径等多个指标进行综合评分,优化柔红霉素纳米粒的制备工艺。②以SD大鼠为实验材料,采用两步滤过法获得微血管段,胶原酶消化,低分子右旋糖苷密度离心获得脑微血管内皮细胞,接种4小时换液使获得的细胞纯化。倒置显微镜下观察细胞的形态,细胞Ⅷ因子相关抗原(ⅧF-Ag)免疫组化法鉴定细胞及其纯度,MTT法检测细胞活性。将传至第2代的脑微血管内皮细胞接种于Transwell内侧,细胞培养一周。通过柔红霉素纳米化在体外模型中的分布情况,探讨柔红霉素纳米粒透过血脑屏障的体外模型通过率。结果:①在优化条件下制备的DAM-PBCA-NP为乳红色胶体溶液,毫微粒外形圆整光滑、分布均匀、不粘连,平均粒径(82±3)nm,包封率为(92.65±1.87)%,载药量为(68.52±1.56)%.②用我们的分离培养方法获得的脑微血管内皮细胞在倒置显微镜下细胞呈多角形或“铺路石”形,单层贴壁生长。培养的细胞Ⅷ因子相关抗原免疫组化试验阳性,细胞纯度为90%.③柔红霉素纳米粒透过血脑屏障的体外模型通过率为(70.15±1.23)%。讨论:①通过优选工艺所制得的柔红霉素纳米粒可以满足本实验研究的需要,该优化条件可作为DAM-PBCA-NP的最佳制备工艺。②我们实验采用的两步滤过获得脑微血管段,胶原酶消化,低分子量右旋糖昔密度离心可分离和培养大鼠脑微血管内皮细胞。这一方法较其他方法程序简单,获得细胞纯度高,适合用于体外BBB模型的建立。③柔红霉素纳米粒透过血脑屏障的体外模型通过率为(70.15±1.23)%,表明对进一步的体内试验及临床试验提供了实验依据。更多还原

【Abstract】 Objective: To prepare nanoparticle drug carriers for daunorbic, and to simulate a simple in-vitro cell model of blood brain barrier, as well as to provide evidence on clinical application of daunorbic nanoparticles.Methods:①Polybutylcyano acrylate was selected to be drug carrier, and daunorubicin polybutylcyano acrylate nanoparticles were prepared using emulsifi-polymerization. DAM-PBCA-NP, examined with single factor experiment and equality test, was analyzed and evaluated on multi factors, in terms of drug load of daunorubicin, coating rate, and average particle diameter, for optimizing preparation of daunorubicin nanoparticle.②SD rats were selected as the experimental material. Micro-blood vessels were attained using two-step filtration. Assimilated by collagen enzyme and centrifuged with low-molecular dextran, micro-blood vessel endothelium cells were gained. Cell culture base was changed 4 hours after inoculation. With inversion microscope, cellular purification was immunohistochemically determined byⅧF-Ag, and cellular activation was examined using MTT method. The second generation micro-blood vessel endothelium cells were inoculated inner Transwell and cultured for 1 week. Rate of permeation through blood brain barrier was determined by DAM-PBCA-NP distribution in the in-vitro model.Results:①DAM-PBCA-NP solution presented red with optimized preparation. Particle shape was round and smooth, well distributed, with an average diameter of (82±3) nm. Its coating rate reached (92.65±1.87)%, and drug load was (68.52±1.56)%.②Micro-blood vessel endothelium cells gained with separated culture approach had multi-angle shape with monolayer growth. Immunohistochemical test showed positive, and cellular purification reached 90%.③Rate of permeation through blood brain barrier was (70.15±1.23) %.Discussion:①The optimized preparation of DAM-PBCA-NP satis fied the needs in the present study, which could be considered the best preparation.②The two-step method for attaining rat micro-blood vess el endothelium cells is easy to approach, and the high cellular purification was suitable for BBB model.③Rate of permeation through blood brain barrier was (70.15±1.23) %, which has provided clinical evidence for further in-vivo experiments.更多还原